Skip to main content

Advertisement

We’d like to understand how you use our websites in order to improve them. Register your interest.
Download PDF

Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells

BMC Medical Genomics volume 12, Article number: 9 (2019) Cite this article

Abstract

Background

It has been found that chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC). CRS can be caused by gastro-oesophageal reflux (GOR) that may reach nasopharynx. The major component of refluxate, bile acid (BA) has been found to be carcinogenic and genotoxic. BA-induced apoptosis has been associated with various cancers. We have previously demonstrated that BA induced apoptosis and gene cleavages in nasopharyngeal epithelial cells. Chromosomal cleavage occurs at the early stage of both apoptosis and chromosome rearrangement. It was suggested that chromosome breaks tend to cluster in the region containing matrix association region/scaffold attachment region (MAR/SAR). This study hypothesised that BA may cause chromosome breaks at MAR/SAR leading to chromosome aberrations in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is a deletion hotspot in NPC.

Methods

Potential MAR/SAR sites were predicted in the AF9 gene by using MAR/SAR prediction tools. Normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) were treated with BA at neutral and acidic pH. Inverse-PCR (IPCR) was used to identify chromosome breaks in SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR). To map the chromosomal breakpoints within the AF9 SAR and non-SAR regions, DNA sequencing was performed.

Results

In the AF9 SAR region, the gene cleavage frequencies of BA-treated NP69 and TWO4 cells were significantly higher than those of untreated control. As for the AF9 non-SAR region, no significant difference in cleavage frequency was detected between untreated and BA-treated cells. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukaemia (MLL) gene in an acute lymphoblastic leukaemia (ALL) patient.

Conclusions

Our findings suggest that MAR/SAR may be involved in defining the positions of chromosomal breakages induced by BA. Our report here, for the first time, unravelled the relation of these BA-induced chromosomal breakages to the AF9 chromatin structure.

Peer Review reports

Background

Genetic alterations [1], epigenetic changes [2], and environmental factors [3] are thought to be involved in the development of nasopharyngeal carcinoma (NPC). Several environmental risk factors that contribute to NPC have been identified. These include Epstein-Barr virus (EBV) infection [4, 5], dietary exposure to nitrosamines [6] as well as occupational exposure to smokes, wood dust, formaldehyde and intense industrial heat [6,7,8]. In addition, prior history of chronic nose and ear diseases (such as chronic rhinitis, sinusitis and otitis media) has also long been recognised as a risk factor for developing NPC [9,10,11,12,13,14,15]. Individuals with chronic rhinosinusitis (CRS), the inflammation of the nose and paranasal sinuses, have been shown to have a significantly higher risk of developing NPC as compared with the control individuals without CRS [15]. Although chronic inflammation of nose or ear has long been recognised as a risk factor for NPC, the underlying mechanisms by which this risk factor may contribute to NPC pathogenesis remain elusive.

Gastro-oesophageal reflux disease (GORD) is one of the major aetiological factors of chronic inflammation of sinonasal tract or ear [16,17,18,19,20]. GORD is caused by the flowing back of gastric duodenal contents into the oesophagus. It has been reported that the gastric duodenal refluxate may flow beyond the oesophagus. In turn, the gastric duodenal contents may affect the tracheobronchopulmonary tree, larynx, pharynx, sinonasal tract and middle ear [18, 21, 22]. The typical GORD symptoms such as heartburn and acid regurgitation may not be present in half of these patients [19]. Thus, these atypical manifestations of GORD are not only referred as extraoesophageal reflux (EOR) or laryngopharyngeal reflux [18, 23] but also as ‘silent reflux’ [19].

GORD is related to various inflammatory disorders. These inflammatory disorders include gastritis [24, 25], oesophagitis [26,27,28], laryngitis [29,30,31], pharyngitis [32, 33], post-nasal drip [34], otitis media [35,36,37,38] and asthma [39,40,41]. Moreover, the relation between CRS and GORD has increasingly received much concern [33, 42, 43]. It has been reported that individual with GORD has a significantly higher risk of developing CRS [44]. The prevalence of acid pharyngeal reflux in patients with CRS has been found to be higher than that in the normal controls (64% vs 18%) [42]. Seventy-eight percent of patients with CRS has been observed to have GOR [45]. Nasopharyngeal reflux has been demonstrated in both paediatric [46,47,48,49] and adult groups [34, 42, 43, 50].

Besides, GORD has also been related to various cancers. These cancers include stomach cancer [51, 52], oesophageal adenocarcinoma [53, 54], laryngeal cancer [55], pharyngeal cancer [56] and lung cancer [57]. Bile acid (BA), the major component of acid refluxate has been identified as a carcinogen in human malignancies (reviewed in [58]). It has been found that the levels of total pepsin and BA in the saliva of patients with laryngopharyngeal reflux were approximately three-fold higher than those of the normal volunteers [59]. It has also been reported that the levels of total pepsin and BA in the saliva of early laryngeal carcinoma patients were about four-fold higher than those of the normal controls [60]. In addition, BA has also been shown to have the carcinogenic effect in human hypopharyngeal squamous carcinoma FaDu cells through epithelial-mesenchymal transition (EMT) [61]. EMT is a major pathway related to cancer invasion and metastasis [62]. These observations suggested a potential role for biliary reflux in the pathogenesis of laryngeal and pharyngeal cancers.

There are strong associations among oxidative stress, inflammation and cancer [63,64,65]. Oxidative stress may activate nuclear factor-kappa B (NF-kappa B) [66] which plays a vital role in inflammatory response [67]. The activation of this transcription factor leads to the expression of genes involved in inflammation [66]. On the other hand, the inflammatory condition generates excessive reactive oxygen species (ROS) in cells. The free radicals may interact directly with DNA or interfere with DNA repair system. These, in turn, elevate the mutation rate in the inflammatory cells. Therefore, chronic inflammation predisposes the cells to neoplastic transformation. Cytokines have been found to be the important mediators that relate inflammation to cancer through oxidative stress [68]. It has been demonstrated that the combination of BA and acid triggered NF-kappa B activation in human hypopharyngeal epithelial cells. This, in turn, leads to overexpression of genes associated with antiapoptosis and oncogenic properties [69]. NF-kappa B pathway is well known to be a proinflammatory signalling pathway. This pathway is mainly activated by proinflammatory cytokines such as interleukin 1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) [70]. ROS are known to act as the messengers in NF-kappa B activation. It has been found that the anti-inflammatory cytokine IL-10 was able to inhibit the NF-kappa B activation in the stimulated macrophages via reduction of ROS [71].

It has recently been reported that the level of BA in the serum of NPC patients was significantly higher than that of the normal controls. The level of BA in the serum of NPC patients significantly inhibited the secretion of the IL-10 protein in CD4+ CD5- T cells [72]. IL-10 is suggested to have an anti-inflammatory role through reduction of oxidative stress induced by the proinflammatory factors. Treatment of Caco-2 intestinal epithelial cells with proinflammatory factors such as TNF-alpha, serotonin, adenosine and melatonin has been shown to induce oxidative damage in proteins and lipids. IL-10 was found to be able to reverse the oxidative damage by restoring the activities of antioxidant enzymes such as catalase, superoxide dismutase and glutathione peroxidise [73]. It has also been demonstrated that IL-10 inhibited hydrogen peroxide (H2O2) generation triggered by interferon (IFN)-gamma or TNF-alpha-activated macrophages [74]. Our previous study provided clear evidence that BA triggered oxidative stress in normal nasopharyngeal epithelial and NPC cells. The effect of BA in the induction of oxidative stress was enhanced by the acid [75]. These findings unravelled a possibility that oxidative stress provoked by the acidic gastric duodenal content may be a vital factor leading to the inflammation-induced carcinogenesis in the nasopharyngeal epithelium. It will be intriguing to investigate the relation between BA and proinflammatory or anti-inflammatory factors in the context of direct exposure of the nasopharyngeal epithelial cells to the refluxate.

In addition, BA-induced apoptosis has been suggested to be a possible mechanism underlying the pathogenesis of Barrett’s oesophagus, oesophagus adenocarcinoma and colon cancer [76,77,78]. Chromosomal cleavage is a hallmark of apoptosis. Initially, the chromosomal DNA is being cleaved and detached from their binding sites on the nuclear scaffold. The release of rosettes and loops of chromatin produces the high-molecular-weight (HMW) DNA of 200 to 300 and 30 to 50 kbp, respectively [79,80,81]. In the later stage of apoptosis, the HMW DNA is further degraded into internucleosomal DNA fragments of 180 to 200 bp [82, 83]. In our previous study, we demonstrated that BA was able to induce apoptosis in normal nasopharyngeal epithelial and NPC cells. We further demonstrated that BA-induced apoptosis resulted in chromosome breakages within the AF9 gene. These chromosome breakages were abolished by the caspase-3 inhibitor. Given that caspase-3 is the primary activator of caspase-activated DNase (CAD), our findings suggested that CAD may play an important role in mediating the chromosomal cleavages during BA-induced apoptosis [75].

It has been observed that the apoptotic nuclease CAD is closely associated with the nuclear matrix in the cells undergoing apoptosis [84]. Chromosomal DNA binds to the nuclear matrix through matrix association region/scaffold attachment region (MAR/SAR) [85]. It is plausible that when CAD cleaves the chromosomal DNA, it potentially cleaves at MAR/SAR. Thus, we hypothesised that BA-induced apoptosis may cause DNA breakages preferentially at MAR/SAR sites leading to chromosome rearrangement in NPC. Our study focuses on the AF9 gene which is located at 9p22 because 9p22 is one of the deletion hotspots in NPC [86]. In the present study, we performed in silico prediction of MAR/SAR within the AF9 gene. We demonstrated that the AF9 gene cleavage frequency within the SAR region was significantly higher in BA-treated cells as compared with the untreated control. By contrast, there was no significant difference in the AF9 gene cleavage frequency within the non-SAR region between BA-treated and untreated control cells. Our results suggest a role for MAR/SAR in defining the positions of chromosomal breakages mediated by BA-induced apoptosis.

Methods

Cell lines and chemicals

NP69 normal nasopharyngeal epithelial cell line was generously provided by Prof. Tsao Sai Wah (The University of Hong Kong, Hong Kong, China) and Prof. Lo Kwok Wai (The Chinese University of Hong Kong, Hong Kong, China). TWO4 NPC cell line was kindly given by Prof. Sam Choon Kook (formerly from University of Malaya, Malaysia).

Keratinocyte-SFM medium (17005–042), RPMI 1640 medium (21870–076), penicillin/streptomycin (15140–122), L-glutamine (25030–081) and fetal bovine serum (10270–098) were bought from GIBCO, Invitrogen, USA. Taurocholic acid sodium salt hydrate (T4009), sodium glycochenodeoxycholate (G0759), glycocholic acid sodium (G2878), sodium deoxycholate (D2510), sodium glycodeoxycholate (G6132), dibasic sodium phosphate (255793) and citric acid (251275) were purchased from Sigma, USA. Ammonium acetate (101116) was procured from Merck, Germany. Chloroform (288306) and isoamyl alcohol (W205702) were purchased from Sigma-Aldrich, Malaysia. Phenol (UN2821) and sodium dodecyl sulfate (SDS) (151–21-3) were obtained from Amresco, USA. Phusion High-Fidelity DNA Polymerase (F-530 L) was bought from Finnzymes, Finland. BamH I (R013S), Kpn I (R0142S), Nde I (R0111S), Hind III (R0104S), Xba I (R0145S), T4 DNA Ligase (M0202 L) and DNA Polymerase I, Large (Klenow) Fragment (M0210S) were procured from New England Biolabs (NEB), USA. dNTP mix (U1515) was purchased from Promega, USA. PCR primers were obtained from First Base Laboratories. QIAquick Gel Extraction Kit (28704) and Nucleotide Removal Kit (28304) were bought from QIAGEN, Germany.

In silico prediction of MAR/SAR within the AF9 gene

MAR/SAR recognition signature (MRS)

The AF9 gene sequence was accessed from Ensembl database [EMBL:ENSG00000171843]. By using DNASTAR software (Lasergene, USA), MAR/SARs within the AF9 gene were predicted by searching MAR/SAR recognition signature (MRS). MRS is a bipartite sequence strongly related to MAR/SAR [87]. MRS consists of two nucleotide motifs that are found within a distance of 200 bp. The first nucleotide motif is an 8 bp degenerate sequence, AATAAYAA. Exact match is required for this 8 bp sequence. The second nucleotide motif is a 16 bp degenerate sequence, AWWRTAANNWWGNNNC, where Y = C or T; W = A or T; R = A or G; N = A, C, G or T. One mismatch is permitted within the 16 bp sequence. The distance between these two degenerate sequences should be within 200 bp. Each sequence can be found on either Watson (W) strand or Crick (C) strand. The sequences may be overlapping or one precedes the other. When there is more than one motif of either 8 or 16 bp to be found within the constraint of the pattern, they are considered as a single MRS. Besides, when there is more than one MRS to be identified within close proximity, they are regarded as a single potential MAR/SAR site. The locations of the MRS predicted MAR/SARs were compared with the locations of the experimentally determined MAR/SARs identified in previous studies [88, 89].

We have also performed in silico prediction of MAR/SAR in the abelson murine leukaemia viral oncogene homolog 1 (ABL) gene by using MRS [90]. We only found one predicted MAR/SAR site which matches to the experimentally determined MAR/SAR in the ABL gene. However, the distance between the 8 bp sequence element and the 16 bp sequence element was found to be 248 bp. Therefore, in this study, we set the maximal distance between 8 bp sequence element and the 16 bp sequence element at 250 bp.

SMARTest and MAR-finder

The AF9 gene sequence was further analysed by using two MAR/SAR prediction tools, namely, SMARTest (http://www.genomatix.de) [91] and MAR-Finder (http://genomecluster.secs.oakland.edu/marwiz/) [92].

Cell cultures

NP69 cells were grown in Keratinocyte-SFM medium supplemented with 2% (v/v) heat-inactivated fetal bovine serum, 4–5 ng/ml recombinant Epidermal Growth Factor (rEGF), 40–50 μg/ml Bovine Pituitary Extract (BPE), 100 U/ml penicillin and 100 μg/ml streptomycin. TWO4 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were cultured with 5% CO2 at 37 °C.

Preparations of BA cocktail and media for BA treatment

The BA cocktail was prepared as previously described [93]. It consists of an equimolar mixture of sodium salts of deoxycholic acid, glycochenodeoxycholic acid, glycocholic acid, glycodeoxycholic acid and taurocholic acid. The concentration of each of the five bile salts was 0.02 mM for a total BA concentration of 0.1 mM. Total BA concentrations in the refluxate of patients with Barrett’s oesophagus were reported to range from 0.03 to 0.82 mM [94]. Higher concentrations of BA (as high as 7.6 mM) have also been observed in the refluxate of some patients with Barrett’s oesophagus [95]. Therefore, in this study, we used a concentration within the physiological range (0.5 mM).

In addition, it has been reported that nasopharyngeal reflux was more prevalent in CRS patients. Nasopharyngeal pH less than 5 has been observed in 76% of these nasopharyngeal reflux-related CRS patients [43]. Similarly, in a 24-h pH monitoring study, abnormal nasopharyngeal pH (nasopharyngeal pH mean was 5.6917) has been reported in patients with GORD related-chronic respiratory diseases (otitis, sinusitis, laryngitis, epiglottitis, recurrent stridor, asthma and recurrent pneumonia). A 5.8 nasopharyngeal pH was regarded as the most sensitive and specific cut-off point to show the presence of abnormal pH-metry in the patients with nasopharyngeal reflux related-chronic respiratory diseases [96]. Therefore, in our studies, the BA treatments were performed at neutral pH (pH 7.4) and acidic pH (pH 5.8). The media used for BA treatment at acidic pH were acidified to pH 5.8 with citrate phosphate buffer.

Nested inverse polymerase chain reaction (IPCR) detection of BA-induced chromosome breaks

BA treatment

NP69 cells (1.5 × 104) and TWO4 cells (2.5 × 104) were seeded in 60 mm culture dishes and allowed to grow for 2 days. NP69 cells were left untreated or treated with 0.5 mM of BA cocktail at pH 7.4 and pH 5.8 for 1 h. TWO4 cells were left untreated or treated with 0.5 mM of BA cocktail at pH 7.4 and pH 5.8 for 3 h.

Genomic DNA extraction

After exposure, the cells were subjected to genomic DNA extraction by using phenol/chloroform/isoamyl alcohol extraction method as previously described [97].

Manipulation of genomic DNA for the AF9 SAR region

In order to prepare the DNA for nested IPCR, several manipulation steps were carried out as previously described [97]. The simplified manipulation steps were illustrated in Additional file 1. Firstly, digestion with 100 U of BamH I (RE1 in Additional file 1) was performed. This was followed by Klenow fill-in, ligation and ethanol precipitation. The DNA was either digested with Kpn I (RE2 in Additional file 1) or Nde I (RE3 in Additional file 1). The digested DNA was cleaned up by using QIAGEN QIAquick Nucleotide Removal Kit according to the manufacturer’s protocol.

Nested IPCR for the AF9 SAR region

The reaction for nested IPCR consisted of 1X of HF buffer (containing 1.5 mM of MgCl2), 0.5 μM of each reverse primer and forward primer, 200 μM of dNTP mix, 0.4 U of Phusion High-Fidelity DNA Polymerase, and 200 ng of DNA template. Sterile ultrapure water was used to replace DNA template in the negative control. The cycle condition used in the first round was as below: 30 s of 98 °C for 1 cycle (initial denaturation), followed by 30 cycles of 98 °C for 10 s (denaturation), 69 °C for 30 s (annealing), 72 °C for 15 s (extension), followed by 1 cycle of 72 °C for 10 min (final extension). Two μl of 5-fold diluted first round IPCR product was used as DNA template for the second round. The cycle condition used in the second round was similar to that in the first round, except that the annealing temperature was 57 °C. The primers used in the first round of IPCR were 5’-ATTCTAGACCCCAAAAAATTCTCAG-3′ (reverse) and 5’-CTCTTAATGCCACTGCCATGA-3′ (forward), whereas the primers used in the second round were 5’-CATATCCTTTTCATACCTGG-3′ (reverse) and 5’-ATTGGTGTCAATCAAATGC-3′ (forward). The IPCR amplifications were performed by using a Veriti 96 Well Thermal Cycler (Applied Biosystems, USA).

Manipulation of genomic DNA and nested IPCR for the AF9 non-SAR region

The manipulation steps were similar with that of the SAR region, except that Hind III (RE2 in Additional file 1) and Xba I (RE3 in Additional file 1) were used for the AF9 non-SAR region instead of Kpn I and Nde I. The cycle condition used in the first round of IPCR was as below: 30 s of 98 °C for 1 cycle (initial denaturation), followed by 30 cycles of 98 °C for 10 s (denaturation), 64 °C for 30 s (annealing), 72 °C for 22 s (extension), followed by 1 cycle of 72 °C for 10 min (final extension). Two μl of 5-fold diluted first round IPCR product was used as DNA template for the second round. The cycle condition of the second round was similar to that of the first round, except that the annealing temperature was 63 °C and the extension time was 15 s. The primers used for the first round of IPCR were 5′- TACCAAACATTTTGAGTCCTACAG-3′ (reverse) and 5′- GGCATTCAGGTGAGTAGTTTATTC-3′ (forward), whereas the primers used in the second round were 5’-AGCAGTAGACTTTTGTAACCTCAC-3′ (reverse) and 5′- AGGGGATGACTTTTCTTCAATC-3′ (forward).

Agarose gel electrophoresis and DNA sequencing of the IPCR bands

To visualise the AF9 cleaved fragments, the IPCR products were loaded on 1% agarose gel and stained with ethidium bromide. The IPCR bands representing the AF9 cleaved fragments were excised. The IPCR products were extracted by using QIAGEN QIAquick Gel Extraction Kit and sequenced. The sequencing results were then annotated by blasting the human genome database (Nucleotide BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi). The breakpoints of the AF9 cleaved fragments were identified by aligning the sequencing data with the AF9 gene sequence retrieved from Ensembl database [EMBL:ENSG00000171843]. This was done by using Seqman DNASTAR software (Lasergene, USA). A genomic map depicting the positions of the chromosome breaks in relation to the chromatin structure was constructed.

Quantification of gene cleavage frequency

The IPCR assays were carried out in two to four sets per experiment. Each set of IPCR assay consisted of three to six replicates per cell sample. The gene cleavage frequencies represent the average number of the AF9 cleaved fragments detected in two to three independent experiments.

Prediction of topo II consensus sites

Topo II consensus sites were predicted as previously described [98, 99]. Topo II consensus site was proposed to be associated with an 18 bp DNA sequence, 5’RNYNNCNNGYNGKTNYNY 3′. There are ten specific and eight non-specific nucleotides in this sequence. One mismatch is allowed for the ten of the specific nucleotides in one DNA strand whereas five mismatches are allowed in the opposite strand.

Statistical analysis

Data were presented as means with standard deviation (SD). Student’s t-test was used to evaluate the significance of differences between the untreated control and the treated groups in flow cytometric analyses and IPCR assays. Mann-Whitney U test was used to evaluate the significance of differences in cleavage frequencies between the SAR region and non-SAR region. All statistical tests were two-sided. Differences were considered statistically significant at p-value < 0.05.

Results

In silico prediction of MAR/SAR

MAR/SAR recognition signature (MRS)

This study targeted the AF9 gene located at 9p22. The AF9 gene is 280,880 bp in length [EMBL:ENSG00000171843]. It consists of 10 exons (Additional file 2). Potential MAR/SAR sites in the AF9 were predicted by MAR/SAR recognition signature (MRS). MRS was proposed to be strongly associated with MAR/SAR [87]. Forty-one MRS predictions were found in the AF9 gene. These 41 MRS correspond to 29 MAR/SAR candidates, as some of the MRSs which cluster within close proximity were regarded as a single potential MAR/SAR site.

Table 1 shows the nucleotide positions of the MRSs with their sequence composition, relative orientation, distance between the two sequence elements and location of the MRSs in the exon or intron of the AF9 gene. Intron 2 with 164 kb in length is the largest intron of the AF9 gene. Almost half of the MAR/SAR sites (14 out of the 29 predicted MAR/SARs) were found in this largest intron (MAR/SARs 2–15 in Table 1). Both intron 3b (MAR/SARs 17–21 in Table 1) and intron 4 (MAR/SARs 22–26 in Table 1) were found to contain five MAR/SAR sites. Two potential MAR/SAR sites (MAR/SARs 27–28 in Table 1) were identified in intron 7. Intron 1 (MAR/SAR 1 in Table 1), intron 3a (MAR/SAR 16 in Table 1) and intron 9 (MAR/SAR 29 in Table 1) were all found to contain one MAR/SAR site.

Table 1 MRS-predicted MAR/SAR sites within the AF9 gene
Full size table

Figure 1 (yellow arrows) shows the distribution of the MRS predicted MAR/SAR sites in the AF9 gene. Two experimentally determined MAR/SARs have been reported in the previous study. These two MAR/SARs were designated as SAR1 and SAR2. SAR1 is located in intron 4. SAR2 spans from exons 5 to 7 [88]. Based on the locations of the previously reported biochemically defined MAR/SAR sites and the presently predicted MAR/SAR sites, a SAR region (contains MAR/SAR) and a non-SAR region (does not contain MAR/SAR) were determined as the region of our study (Fig. 1). The AF9 SAR region contains one MRS- predicted MAR/SAR site (MAR/SAR 24) which matches to the biochemically defined SAR1. By contrast, the AF9 non-SAR region is a region which does not contain any biochemically extracted MAR/SAR or MRS- predicted MAR/SAR.

Fig. 1
figure1

Potential MAR/SAR sites within the AF9 gene. The AF9 genomic map from nucleotide positions 601–281,480 is depicted [EMBL:ENSG00000171843]. The locations of exons 1 to 10 and BamH I (B) restriction sites are shown. Green boxes represent the two previously identified patient BCRs which were indicated as BCR1 and BCR2 [88]. Yellow boxes represent the two experimentally verified MAR/SARs (denominated as SAR1 and SAR2) reported in the previous study [88]. Yellow, blue and red arrows represent the potential MAR/SAR sites predicted in our present study by using MRS, SMARTest and MAR-Finder, respectively. Based on the previous reports and the in silico prediction in the present study, a SAR region (contains MAR/SAR) and a non-SAR region (does not contain MAR/SAR) were determined to be the regions of study

Full size image

SMARTest and MAR-finder

The AF9 sequence was further analysed by using two MAR/SAR prediction programs, namely SMARTest (http://www.genomatix.de) and MAR-Finder (http://genomecluster.secs.oakland.edu/marwiz/). The distribution of MAR/SARs predicted by SMARTest and MAR-Finder were shown in Fig. 1 (blue and red arrows, respectively). Table 2 shows the nucleotide position of the potential MAR/SAR sites identified by SMARTest. There were 51 MAR/SARs predicted within the AF9 gene. There was no MAR/SAR predicted within the non-SAR region. The closest MAR/SAR at its 5′ site locates at coordinates 66,686–67,255 (MAR/SAR 14 in Table 2) while the closest MAR/SAR at its 3′ site locates at coordinates 85,486–85,800 (MAR/SAR 15 in Table 2). On the other hand, there were four MAR/SARs predicted within the SAR region. These four MAR/SARs locate at coordinates 237,321–237,855, 240,926–241,315, 244,311–244,710 and 245,416–245,850 (MAR/SARs 46–49 in Table 2). The locations of these four SMARTest- predicted MAR/SARs match to the experimentally determined SAR1 (Fig. 1).

Table 2 SMARTest-predicted MAR/SAR sites within the AF9 gene
Full size table

Figures 2 a, b and c show the MAR-Finder predicted MAR/SARs within the AF9 gene for coordinates 0–100,000, 100,000–200,000 and 200,000–282,080, respectively [Ensembl:ENSG00000171843]. MAR-Finder predicted seven MAR/SAR sites within the AF9 gene. These seven potential MAR/SARs locate at 57200 (Fig. 2 a), 124,700, 125,200, 195,000, 197,000 (Fig. 2 b), 205,900 and 280,000 (Fig. 2 c). There was no MAR/SAR predicted within the SAR region or the non-SAR region.

Fig. 2
figure2

MAR-Finder predictions within the AF9 gene. (a) Coordinates 0–100,000 (b) Coordinates 100,000–200,000 (c) Coordinates 200,000–282,080 [Ensembl:ENSG00000171843]. There were seven MAR/SARs predicted within the AF9 gene. These seven potential MAR/SARs locate at 57200 (a), 124,700, 125,200, 195,000, 197,000 (b), 205,900 and 280,000 (c)

Full size image

IPCR detection of chromosome breaks within the AF9 SAR and non-SAR regions upon BA treatment

Nested IPCR was employed to investigate if BA-induced apoptosis does lead to cleavage in the AF9 SAR and non-SAR regions. Genomic DNA extraction and subsequent IPCR were performed on BA-treated NP69 and TWO4 cells. Based on the primers position, if there is no chromosomal breakage detected, the IPCR product for the SAR region of the AF9 gene will be 944 bp (~ 950 bp), while for the non-SAR region of the AF9 gene, the IPCR product will be 956 bp (~ 950 bp). If there is any chromosomal breakage detected, for both SAR and non-SAR regions, IPCR products of less than 950 bp will be obtained.

For the SAR region, NP69 cells treated with BA at pH 7.4 (Lanes 6–9) and pH 5.8 (Lanes 13–15) showed numerous IPCR bands of less than 950 bp (Fig. 3a i). These bands represent the AF9 gene cleavages within the SAR region. Two IPCR bands were also detected in the untreated NP69 cells (Lanes 3 and 4). By using flow cytometric analyses, we have demonstrated that untreated control contained a small percentage of apoptotic cells [75]. The background might be contributed by endogenous DNA breaks which occur in these dying cells. As shown in Fig. 3b left columns, the AF9 gene cleavage frequency detected in NP69 cells treated with BA at pH 7.4 was approximately 3.9-fold higher than that in untreated NP69 cells (p-value = 0.015). It was 4.9-fold higher than that of untreated control in cells exposed to BA at pH 5.8 (p-value = 0.032).

Fig. 3
figure3

Identification of chromosome breaks in BA-treated NP69 cells. IPCR was employed to identify the AF9 gene cleavages in NP69 cells after exposed to BA. a Representative gel picture showing the AF9 gene cleavages identified by IPCR within: (a i) SAR region (a ii) Non-SAR region. NP69 cells were left untreated (a i, Lanes 1–5; a ii, Lanes 1–6) or treated for 1 h with 0.5 mM of BA at pH 7.4 (a i, Lanes 6–10; a ii, Lanes 7–12) and pH 5.8 (a i, Lanes 11–15; a ii, Lanes 13–18). Genomic DNA extraction and nested IPCR were performed as described in “Methods” section. The side bracket represents the IPCR bands derived from the AF9 cleaved fragments. M: 100 bp DNA marker. N: negative control for IPCR. b The average number of the AF9 gene cleavages identified in BA-treated NP69 cells. Data are expressed as means and SDs of two independent experiments. Each experiment consisted of two to four sets of IPCR carried out in three to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p < 0.05, ** p < 0.01. NS, no significant difference

Full size image

As for the non-SAR region, untreated NP69 cells (Lanes 1–6), and NP69 cells treated with BA at pH 7.4 (Lanes 7–10 and 12) and pH 5.8 (Lanes 13–18) showed numerous IPCR bands of less than 950 bp (Fig. 3a ii). These bands represent the AF9 gene cleavages within the non-SAR region. There was no significant difference in the cleavage frequency between untreated NP69 cells and NP69 cells treated with BA at pH 7.4 (p-value = 0.807) or pH 5.8 (p-value = 0.086) (Fig. 3b right columns).

Similar results were observed in TWO4 cells. Numerous IPCR bands of less than 950 bp were detected in TWO4 cells after treatment with BA at pH 7.4 (Fig. 4a i, Lanes 8–12) and pH 5.8 (Fig. 4a i, Lanes 13–18). These bands represent cleaved AF9 gene within the SAR region. A few IPCR bands were also detected in the untreated TWO4 cells (Fig. 4a i, Lanes 1–3) which might be due to spontaneous DNA breaks. The AF9 gene cleavage frequencies of TWO4 cells treated with 0.5 mM of BA at neutral pH and acidic pH are 1.8-fold (p-value = 0.004) and 1.6-fold (p-value = 0.036) higher than that of the untreated control, respectively (Fig. 4b left columns).

Fig. 4
figure4

Identification of chromosome breaks in BA-treated TWO4 cells. Genomic DNA was extracted from BA-treated TWO4 cells for nested IPCR as described in “Methods” section. a Representative gel picture showing the AF9 gene cleavages in BA-treated TWO4 cells detected within: (a i) SAR region (a ii) Non-SAR region. TWO4 cells were left untreated (Lanes 1–6) or treated for 3 h with 0.5 mM of BA at pH 7.4 (Lanes 7–12) and pH 5.8 (Lanes 13–18). The IPCR bands derived from the AF9 cleaved fragments were indicated by the side bracket. M: 100 bp DNA ladder. N: Negative control for IPCR. b The average number of AF9 gene cleavages detected by IPCR. Data represents means and SDs of three independent experiments. Each experiment consisted of at least two sets of IPCR assays performed in five to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p < 0.05, ** p < 0.01. NS, no significant difference

Full size image

As for the non-SAR region, numerous IPCR bands of less than 950 bp were detected in untreated TWO4 cells (Fig. 4a ii, Lanes 1–6), and TWO4 cells treated with BA at pH 7.4 (Fig. 4a ii, Lanes 7–12) and pH 5.8 (Fig. 4a ii, Lanes 13–16 and 18). These bands represent cleaved AF9 gene within the non-SAR region. As summarised in the bar chart in Fig. 4b (right columns), there was no significant difference in the cleavage frequency of the AF9 non-SAR region between untreated TWO4 cells and TWO4 cells treated with BA at pH 7.4 (p-value = 0.903) or pH 5.8 (p-value = 0.429).

These findings clearly demonstrate that MAR/SAR mediates the gene cleavages in BA-induced apoptosis in NP69 and TWO4 cells at both neutral and acidic pH. However, in both NP69 and TWO4 cells, there were obviously more cleavage bands detected in the non-SAR region (Figs. 3a ii and 4a ii) as compared with the SAR region (Figs. 3a i and 4a i). Table 3 shows the differences in the cleavage frequencies between SAR and non-SAR regions. The cleavage frequencies of the non-SAR region detected in untreated NP69 and TWO4 cells were 4.0-fold (p-value = 0.001) and 5.7-fold (p-value < 0.001) higher than those of the SAR region, respectively. It is possible that other chromatin structures may have contributed to DNA fragility of the AF9 non-SAR region. In addition to MAR/SAR sequence, repeat elements and topoisomerase II (topo II) cleavage site have also been well implicated in mediating chromosome breaks [88, 89, 100]. Hence, this prompted us to investigate the possibility of repeat elements and topo II cleavage site in contributing to DNA fragility of the AF9 non-SAR region.

Table 3 Cleavage frequencies detected in the AF9 SAR and non-SAR regions
Full size table

Identification of repeat elements

By using CENSOR program (https://www.girinst.org/censor/), repeat elements were identified in the AF9 gene. Tables 5 and 6 show the repeat elements identified within the SAR and non-SAR regions, respectively. Eighteen repeat elements were identified within the 10.2 kb SAR region (Table 4). The overall content of repeat elements in the SAR region is 13.81%. Only one out of these 18 repeat elements is found within the amplified region. ERE2_EH (coordinates 245,627–245,728, 102 bp in length) is the only one repeat element identified within the amplified region. This repeat element occupies 11% (102 bp) of the amplified SAR region (944 bp).

Table 4 Repeat elements identified in the AF9 SAR region by CENSOR program
Full size table

On the contrary, nine repeat elements were identified within the 4.2 kb non-SAR region (Table 5). The overall content of repeat elements in the non-SAR region is 41.37%. Three out of these nine repeat elements are found within the amplified region. The three repeat elements identified in this region were two CHARLIE5 (coordinates 74,895–74,998, 104 bp in length and coordinates 75,006–75,169, 164 bp in length) and one AluJr (coordinates 75,192–75,466, 275 bp in length). These three repeat elements take up 57% (543 bp) of the amplified non-SAR region (956 bp). The locations of repeat elements identified within the SAR and non-SAR regions are illustrated in Fig. 5.

Table 5 Repeat elements identified in the AF9 non-SAR region by CENSOR program
Full size table
Fig. 5
figure5

The repeats and topo II sites identified within the SAR and non-SAR regions. a The SAR region. The SAR region that is bordered by the two BamH I sites is 10.2 kb in length (from coordinates 236,059 to 246,292). Green box represents the previously identified patient BCR which is indicated as BCR1. Yellow box shows the previously experimentally isolated MAR/SAR which is indicated as SAR1 [88]. Yellow and blue arrows represent the potential MAR/SARs predicted in this study by using MRS and SMARTest, respectively. Orange arrows represent the predicted topo II consensus sites. Green arrows represent the primers (R1, AF9 236,451 R and F1, AF9 245,385 F) used in the first round of nested IPCR while the purple arrows represent the primers (R2, AF9 236,211 R and F2, AF9 245,507 F) used in the second round of nested IPCR. Black boxes represent the repeat elements. BamH I (B), Kpn I (K) and Nde I (N) restriction sites are shown. b The non-SAR region. The non-SAR region which is bordered by two BamH I sites is 4.2 kb in length (from coordinates 71,116 to 75,277). Orange arrow represents the predicted topo II consensus site. Green arrows represent the primers (R1, AF9 71,653 R and F1, AF9 74,399 F) used in the first round of nested IPCR while the blue arrows represent the primers (R2, AF9 71,282 R and F2, AF9 74,494 F) used in the second round of nested IPCR. Black boxes represent the repeat elements. BamH I (B), Hind III (H) and Xba I (X) restriction sites are shown

Full size image

Prediction of topoisomerase II consensus sites

Table 6 and Fig. 5 summarise the topo II consensus sites predicted in the AF9 SAR and non-SAR regions. We predicted eight topo II consensus sites in the AF9 SAR region. The overall content of topo II sites in the SAR region is 1.41%. Two topo II consensus sites fall within the breakpoint cluster region, BCR1. Five topo II consensus sites were found within the biochemically identified MAR/SAR, SAR1. One topo II site was identified next to SAR1. Two out of the eight topo II consensus sites were found within the amplified region. These two topo II consensus sites occupy 3.81% of the amplified region. In contrast to the SAR region, only one topo II consensus site was predicted within the AF9 non-SAR region. The overall content of topo II sites in the non-SAR region is 0.43%. This topo II consensus site is not located within the amplified region.

Table 6 Topo II consensus sites predicted in the AF9 SAR and non-SAR regions
Full size table

Sequencing results

To ascertain that the cleavage fragments identified in IPCR were derived from the cleaved AF9 gene, some of the IPCR bands were excised, purified and sequenced. The sequencing results show that these fragments were all derived from the cleaved AF9 gene. The breakpoints identified within the AF9 SAR region in BA-treated NP69 and TWO4 are shown in Tables 7 and 8, respectively. Intriguingly, several breakpoints (at coordinates 245,509, 245,527, 245,575, 245,577, 245,594, 245,596 and 245,612) were mapped within the AF9 region (at coordinates 245,252–245,612) which was previously reported to be involved in t(9;11)(p22;q23). This chromosome translocation resulted in the formation of mixed lineage leukaemia (MLL)-AF9 fusion gene in acute lymphoblastic leukaemia (ALL) patient [GenBank:AM050804]. It is noteworthy that one of the presently identified breakpoints is identical with that identified in the ALL patient (at coordinate 245,612) [GenBank:AM050804].

Table 7 Breakpoints detected within the AF9 SAR region in BA-treated NP69 cells
Full size table
Table 8 Breakpoints detected within the AF9 SAR region in BA-treated TWO4 cells
Full size table

A breakpoint (at coordinate 245,596) was simultaneously mapped in both NP69 and TWO4 cells treated with BA at neutral pH. Another breakpoint (at coordinate 245,803) was simultaneously detected in TWO4 cells treated with BA at both neutral and acidic pH. Three breakpoints (at coordinates 245,594, 245,596 and 246,116) are similar with those reported in cultured normal blood cells (at coordinate 245,593) and CEM cells (at coordinate 246,114) exposed to etoposide (VP16) [101]. A few breakpoints (at coordinates 245,594, 245,596, 245,664, 245,699, 245,708, 245,803, 245,994, 246,006 and 246,116) are similar with those identified in H2O2-treated NP69 cells (at coordinates 245,591, 245,659, 245,703, 245,796, 246,000 and 246,113) and HK1 cells (at coordinate 245,590) reported in our previous study [97]. In addition, a few chromosome breaks (at coordinates 245,637, 245,649, 245,664, 245,699, 245,708, 245,711, 245,721 and 245,725) fall within a repeat ERE2_EH (at coordinates 245,627–245,728). A map representing the positions of BA-induced chromosome breaks in NP69 and TWO4 cells within the AF9 SAR region is illustrated in Figs. 6 and 7, respectively.

Fig. 6
figure6

Positions of chromosome breaks within the SAR region in BA-treated NP69 cells. The genomic map of AF9 SAR region from nucleotide positions 236,059–246,292 is illustrated above [EMBL:ENSG00000171843]. BamH I (B), Kpn I (K) and Nde I (N) restriction sites are shown. Green arrows represent the primers (R1, AF9 236,451 R and F1, AF9 245,385 F) used in the first round of nested IPCR while the blue arrows represent the primers (R2, AF9 236,211 R and F2, AF9 245,507 F) used in the second round of nested IPCR. Green box represents the previously reported patient BCR which is indicated as BCR1 [88]. Yellow box shows the experimentally determined MAR/SAR which is indicated as SAR1 [88]. Yellow arrows represent the potential MAR/SARs predicted in this study. Blue box represents the AF9 region (at coordinates 245,252–245,612) previously reported to translocate with the MLL gene resulting in the MLL-AF9 fusion gene identified in an ALL patient [GenBank:AM050804]. Black boxes represent repeat elements. Red and green vertical lines represent the presently detected breakpoints in TWO4 cells treated with BA at neutral and acidic pH, respectively. All the chromosome breaks were mapped within BCR1 in close proximity to SAR1. Three chromosome breaks (at coordinates 245,527, 245,575 and 245,596) fall within the AF9 region previously reported to be involved in t(9;11)(p22;q23) in the ALL patient. Five chromosome breaks (at coordinates 245,649, 245,699, 245,708, 245,721 and 245,725) fall within a repeat ERE2_EH (at coordinates 245,627–245,728)

Full size image
Fig. 7
figure7

Positions of chromosome breaks within the SAR region in BA-treated TWO4 cells. The genomic map of AF9 SAR region from nucleotide positions 236,059–246,292 is illustrated above [EMBL:ENSG00000171843]. BamH I (B), Kpn I (K) and Nde I (N) restriction sites are shown. Green arrows represent the primers (R1, AF9 236,451 R and F1, AF9 245,385 F) used in the first round of nested IPCR while the blue arrows represent the primers (R2, AF9 236,211 R and F2, AF9 245,507 F) used in the second round of nested IPCR. Green box represents the previously reported patient BCR which is indicated as BCR1 [88]. Yellow box shows the experimentally determined MAR/SAR which is indicated as SAR1 [88]. Yellow arrows represent the potential MAR/SARs predicted in this study. Blue box represents the AF9 region (at coordinates 245,252–245,612) previously reported to translocate with the MLL gene resulting in the MLL-AF9 fusion gene identified in an ALL patient [GenBank:AM050804]. Black boxes represent repeat elements. Red and green vertical lines represent the presently detected breakpoints in TWO4 cells treated with BA at neutral and acidic pH, respectively. All the chromosome breaks were mapped within BCR1 in close proximity to SAR1. Five chromosome breaks (at coordinates 245,509, 245,577, 245,594, 245,596 and 245,612) fall within the AF9 region previously reported to be involved in t(9;11)(p22;q23) in the ALL patient. One of those breakpoints is identical with that previously identified in the ALL patient (at coordinate 245,612) [GenBank:AM050804]. Three chromosome breaks (at coordinates 245,637, 245,664 and 245,711) fall within a repeat ERE2_EH (at coordinates 245,627–245,728). Two chromosome breaks fall at the same nucleotide position (at coordinate 245,803)

Full size image

Tables 9 and 10 show the breakpoints identified within the AF9 non-SAR region in BA-treated NP69 and TWO4 cells, respectively. One breakpoint was simultaneously detected in two different IPCR replicates which were from two different sets of IPCR derived from NP69 treated with BA at acidic pH. Six chromosome breaks (at coordinates 74,908, 74,914, 74,928, 74,953 and 74,987) fall within the first repeat CHARLIE5 (at coordinates 74,895–74,998). Four chromosome breaks (at coordinates 75,013, 75,034, 75,043 and 75,081) fall within the second repeat CHARLIE5 (at coordinates 75,006–75,169). The genomic maps representing the positions of chromosome breaks within the AF9 non-SAR region detected in NP69 and TWO4 cells are elucidated in Figs. 8 and 9, respectively.

Table 9 Breakpoints detected within the AF9 non-SAR region in BA-treated NP69 cells
Full size table
Table 10 Breakpoints detected within the AF9 non-SAR region in BA-treated TWO4 cells
Full size table
Fig. 8
figure8

Positions of chromosome breaks within the non-SAR region in BA-treated NP69 cells. The genomic map of AF9 non-SAR region from nucleotide positions 71,116–75,277 is illustrated above [EMBL: ENSG00000171843]. BamH I (B), Hind III (H) and Xba I (X) restriction sites are shown. Green arrows represent the primers (R1, AF9 71,653 R and F1, AF9 74,399 F) used in the first round of nested IPCR while blue arrows represent the primers (R2, AF9 71,282 R and F2, AF9 74,494 F) used in the second round of nested IPCR. Black boxes represent the repeat elements. Red and green vertical lines represent the presently identified breakpoints in NP69 cells upon BA treatment at neutral and acidic pH, respectively. One chromosome break (at coordinate 74,928) falls within the first repeat CHARLIE5 (at coordinates 74,895–74,998). Three chromosome breaks (at coordinates 75,013, 75,034 and 75,081) fall within the second repeat CHARLIE5 (at coordinates 75,006–75,169). Two chromosome breaks fall at the same nucleotide position (at coordinate 74,636)

Full size image
Fig. 9
figure9

Positions of chromosome breaks within the non-SAR region in BA-treated TWO4 cells. The genomic map of AF9 non-SAR region from nucleotide positions 71,116–75,277 is illustrated above [EMBL: ENSG00000171843]. BamH I (B), Hind III (H) and Xba I (X) restriction sites are shown. Green arrows represent the primers (R1, AF9 71,653 R and F1, AF9 74,399 F) used in the first round of nested IPCR while blue arrows represent the primers (R2, AF9 71,282 R and F2, AF9 74,494 F) used in the second round of nested IPCR. Black boxes represent the repeat elements. Red and green vertical lines represent the presently detected breakpoints in TWO4 cells upon BA treatment at neutral and acidic pH, respectively. Five chromosome breaks (at coordinates 74,908, 74,914, 74,953, 74,985 and 74,987) fall within the first repeat CHARLIE5 (at coordinates 74,895–74,998). One chromosome break (at coordinate 75,043) falls within the second repeat CHARLIE5 (at coordinates 75,006–75,169)

Full size image

Discussion

Lately, the association between chronic inflammation of sinonasal tract and NPC has increasingly received much attention [15]. One of the major risk factors for the development of CRS is GORD [42, 43, 102]. It has been demonstrated that gastric duodenal refluxate can reach the larynx, pharynx, oral cavity, nasopharynx, nose, sinus, eustachian tube and middle ear. Repeated exposure to gastric duodenal content may result in localised inflammation of these regions [18, 20, 43, 103,104,105,106]. More recently, BA has been shown to cause cell injury and inflammation in the airway epithelium. Treatment of the immortalised human bronchial epithelial cells (BEAS-2B) with BA resulted in increased activity of proinflammatory cytokines (interleukin-8 and interleukin-6) [107]. The airways do not have intrinsic protective mechanisms as found in the oesophagus. Therefore, the tissues of airways are more vulnerable to acid-peptic injury as compared with the oesophagus. Due to this reason, it is conceivable that when the tissues of airways are repeatedly exposed to the gastric duodenal refluxate, the genotoxicity and mutagenicity of gastric duodenal content may also contribute to carcinogenesis in the airways [108].

By using flow cytometric analyses of phosphatidylserine (PS) externalisation and mitochondrial membrane potential (MMP) disruption, we have previously demonstrated that BA induced apoptosis in normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) [75]. We further demonstrated that BA-induced apoptosis triggered oxidative stress and caspase activity. These events, in turn, resulted in cleavages within the AF9 BCR. These cleavages were abolished by the caspase inhibitor, suggesting that these cleavages were mediated CAD. Our findings suggested that one of the potential mechanisms contributing to chromosome rearrangement in NPC could be BA-induced apoptosis, where CAD may be involved [75]. In the present report, we intended to investigate the relation between the positions of BA-induced chromosomal cleavages and the chromatin structure.

It has been known that the BCRs of the AF9 and MLL genes share similar structural elements. These structural elements include MAR/SAR, topo II cleavage site and DNase I hypersensitive site. The similarity in the structural features in the BCRs of the AF9 and MLL genes has been suggested to serve as recombination hot spots leading to the formation of the MLL-AF9 fusion gene in leukaemogenesis [88]. MAR/SARs are DNA sequences that are responsible for chromosomal loop attachment [109]. Topo II cleavage site and DNase I hypersensitive site frequently co-localise with MAR/SAR [109,110,111]. Therefore, we attempted to study the role of MAR/SAR in determining the positions of chromosomal cleavages in BA-induced apoptosis.

The targeted gene in this study was the AF9 gene located on the short arm of chromosome 9 at position 9p22, a common deletion region in NPC. The AF9 gene frequently translocates with the MLL gene at 11q23 resulting in the reciprocal translocation t(9;11)(p22;q23) in leukaemia [88]. The fusion of these two genes has been found to occur predominantly among the patients with de novo acute myelogenous leukaemia (AML). The MLL-AF9 fusion gene was less commonly observed in patients with acute lymphocytic leukaemia (ALL), with myelodysplastic syndrome (MDS) and with therapy-related AML (t-AML) [88, 112].

In the present study, possible MAR/SAR sites in the AF9 gene were predicted by using MRS. MRS is a bipartite sequence element which has been strongly associated with MAR/SARs. MRS is composed of two individual sequence elements which are found together within a distance of approximately 200 bp. However, when the DNA is wrapped around the histones, these two sequence elements exist at a location near the dyad axis of the nucleosome. Hence, they are found parallel to each other in MAR/SAR when the nucleosomes are positioned. This allows them to generate a protein binding site in MAR/SAR. van Drunen and co-workers have analysed more than 300 kb of DNA sequences from several eukaryotic organisms by using MRS. Their studies reported that all the MRS predictions were associated with the experimentally determined MAR/SARs [87]. MRS has been widely used in other studies and proved to be reliable [113,114,115].

The MRS predictions obtained in the present study were compared with the location of experimentally determined MAR/SARs reported in the previous studies [88, 89]. Strissel and co-workers have analysed exons 4 to 10 of the AF9 gene for MAR/SAR. In this region of 61 kb in length, two biochemically extracted MAR/SARs have been reported. These two MAR/SARs were indicated as SAR1 and SAR2. SAR1 is a 6.2 kb MAR/SAR found in intron 4. SAR2 is a 4.6 kb MAR/SAR spans through parts of introns 5 to 7 [88]. To the best of our knowledge, there is no previous report on the experimentally determined MAR/SAR for the AF9 region from exon 1 to intron 3. Four MRS predictions (MAR/SARs 24–1 to 24–4 in Fig. 1) are associated with SAR1. One of these four MRSs was located in a region < 1 kb centromeric to SAR1 (MAR/SAR 24–1 in Fig. 1), whereas the other three MRSs were found within SAR1 (MAR/SARs 24–2 to 24–4 in Fig. 1). One MRS prediction (MAR 27 in Fig. 1) correlates with SAR2. This MRS was found in a region < 1.5 kb telomeric to SAR2. Interestingly, all the MRS-predicted MAR/SARs were found in the introns of the AF9 gene. These results are consistent with those of the other studies which found that MAR/SARs occur more frequently in introns than in exons. This has been previously confirmed by both the experimental extraction [116, 117] and computational prediction [91].

Based on the in silico prediction and the previous studies which reported the experimentally determined MAR/SARs [88], the SAR and non-SAR regions were determined (Fig. 1). A study by van Drunen and colleagues (1999) showed that they never found a MRS which did not correlate with an experimentally verified MAR/SAR. However, their studies also revealed that not all biochemically defined MAR/SARs contain a MRS. Their findings suggested that there is at least a distinct type of MAR/SAR which does not contain a MRS [87]. Thus, in order to investigate if the region which was considered as a non-SAR region contains another type of MAR/SAR which was not predicted by MRS, we further analysed the AF9 sequence by predicting the presence of MAR/SAR using MAR-Finder and SMARTest.

The MAR/SAR analysis rules utilised in these two programs are different from the criteria used in the MAR/SAR prediction by MRS. MAR-Finder utilises statistical inference to predict the occurrence of MAR/SARs. MAR-Finder was developed by using the formulation of a set of biological rules based on the correlation of MAR/SAR with various DNA sequence motifs. These motifs include the TG-rich sequences, origin of replication (ORI), kinked DNA, curved DNA, AT-rich sequences and topo II sites. MAR-Finder has been shown to successfully identify MAR/SAR sites which correlate with those experimentally verified in the human beta-globin gene, PRM1-PRM2-TNP2 domains and human apolipoprotein B locus [92]. By contrast, SMARTest predicts MAR/SARs based on a density analysis of MAR/SAR-associated features described by a weight matrix library [91]. This MAR/SAR matrix library was mainly derived from the following MAR/SAR-associated patterns. Firstly, MAR/SARs have a minimum sequence length of 200–300 bp [118]. Secondly, MAR/SAR sequences are AT-rich [117]. Thirdly, MAR/SARs are associated with a few motifs. These motifs include ATTA, ATTTA, AATATT, ATATTT and AATATATTT [85, 118,119,120,121].

In order to evaluate the capability of SMARTest, Frisch and co-workers analysed six genomic sequences (three human sequences and three plant sequences) with a total of 310 kb in length by using SMARTest. These six genomic sequences contain a total of 37 experimentally determined MAR/SARs. Their studies showed that 19 of 28 SMARTest predictions were true positives (specificity = 68%). These 19 true positives only correlate with 14 of 37 biochemically extracted MAR/SARs in these genomic sequences (sensitivity = 38%), as some of the MAR/SARs contain more than one SMARTest predictions. For comparison, the authors analysed the same six sequences by using MAR-Finder. Twenty of 25 MAR-Finder predictions were true positives (specificity = 80%). These 20 true positives only correlate with 12 of 37 biochemically extracted MAR/SARs in these sequences, as some of the MAR/SARs contain more than one MAR-Finder predictions (sensitivity = 32%) [91]. Given that the MAR/SAR matrix library utilised by SMARTest was derived from AT-rich MAR/SAR, other MAR/SAR classes distinct from AT-rich class were not predicted by SMARTest. However, these MAR/SAR classes distinct from AT-rich class were found by MAR-Finder. Frisch and co-workers further found that some of the experimentally determined MAR/SARs which were not identified by MAR-Finder were detected by SMARTest. Hence, SMARTest and MAR-Finder were suggested to mutually complete each other in MAR/SAR prediction [91].

Due to a lack of report on the experimentally determined MAR/SARs for the AF9 region from exon 1 to intron 3 (approximately 220 kb in length), the sensitivity and specificity of MRS, SMARTest and MAR-Finder were unable to be compared in this study. Nevertheless, the comparison of accuracy among these prediction tools was not the aim of this study. The main purpose of using these different MAR/SAR prediction tools was to predict the MAR/SARs of different classes. By using MRS, SMARTest and MAR-Finder for MAR/SAR prediction, our findings suggested that the non-SAR region does not contain any MAR/SAR (Fig. 1).

Given that chromosomal cleavage is an initial event in both apoptosis and chromosome rearrangements, we employed nested IPCR to identify the chromosome breaks mediated by BA-induced apoptosis. Our findings showed that, for the SAR region, the cleavage frequencies in BA-treated cells were significantly higher than those in the untreated control. On the contrary, for the non-SAR region, there was no significant difference in the cleavage frequencies between the BA-treated cells and untreated control cells. These observations were true for both NP69 and TWO cell lines. However, in both untreated NP69 and TWO4 cells, the cleavage frequencies of the non-SAR region were significantly higher than those of the SAR region. By using CENSOR program, we found that the overall content of repeat elements in the non-SAR region is 3.0-fold higher (41.37% vs 13.81%) than that in the SAR region. Considering that no significant difference in the cleavage frequencies of the non-SAR region was found between the untreated and BA-treated cells, it seems not unlikely that the chromosome breaks in the non-SAR region were not mediated by BA-induced apoptosis. Rather, the chromosome breaks detected in this region were mostly spontaneous breaks due to DNA fragility contributed by these repeat elements. It is likely that repeat elements make the chromosome to be more prone to cleavage. These results are consistent with those of the other studies which reported a high proportion of repeat elements in common fragile sites, including FRA3B, FRA7G, FRA7H, FRA16D and FRAXB. These repeat elements include interspersed repeat elements, long terminal repeats (LTR), transposable elements, Mirs, L1 elements, L2 elements and Alu elements [122, 123]. Therefore, we concluded that MAR/SAR may play an essential role in mediating the gene cleavages in BA-induced apoptosis in NP69 and TWO4 cells at both neutral and acidic pH.

Knowing that topo II was involved in mediating illegitimate recombination [124, 125], we further analysed the SAR and non-SAR regions for topo II consensus sites. The topo II consensus sites were predicted by using an 18 bp consensus sequence [98, 99]. Our findings showed that the proportion of topo II sites in the SAR region was approximately 3-fold higher than that in the non-SAR region (1.41% vs 0.43%). These results seemed to reaffirm the findings of those studies which unravelled that MAR/SARs are the dominant sites for topo II binding and cleavage [109, 126].

In an in vitro system, topo II has been demonstrated to play a critical role in mediating DNA cleavages at acidic pH. Topo II has also been shown to be involved in mediating mutation and cytotoxicity induced by acidic pH in tissue culture models. These findings suggested that topo II-mediated DNA damage may lead to the development of cancers associated with gastro-oesophageal acid reflux [127]. In addition, previous studies have demonstrated that topo II is responsible for chromosomal loops excision in the early stage of apoptosis induced by oxidative stress. This initial event was followed by activation of nucleases leading to degradation of chromosomal DNA into nucleosomal DNA [128].

Our previous study has shown that BA and/or acidic pH induced apoptosis via oxidative stress in nasopharyngeal epithelial cells. In BA-induced apoptosis, we demonstrated that DNA cleavages within the SAR region occurred in a caspase-3-dependent manner, suggesting that CAD is responsible for these DNA cleavages [75]. Besides, we have also previously demonstrated that CAD cleaves the DNA preferentially at MAR/SAR sites during oxidative stress [97]. It has been observed that CAD was closely associated with the nuclear matrix of apoptotic cells [84]. It is conceivable that when CAD binds to the nuclear matrix during apoptosis, CAD preferentially cleaves the DNA at MAR/SAR sequences. It is possible that in BA and/or acidic pH-induced apoptosis, which involves oxidative stress, both topo II and CAD do play a role in mediating the DNA cleavages. The former may take part in mediating the cleavage of loop-sized DNA into HMW fragments whereas the latter may involve in mediating the degradation of chromosomal DNA into nucleosomal DNA. Therefore, our current findings which revealed that BA-induced apoptosis resulted in DNA cleavages within the SAR region may be explained by the close relation among topo II, CAD and MAR/SAR.

Sequencing of IPCR bands detected in the SAR region showed the positions of chromosome breaks within the AF9 BCR1 mediated by BA-induced apoptosis. The AF9 BCR1 is bordered by SAR1 and SAR2 [88, 89]. It is noteworthy that the positions of the chromosome breaks identified in the present study were highly similar to those previously reported in leukaemia patients. A few chromosome breaks were mapped within the AF9 region that was previously reported to translocate with the MLL gene in an ALL patient. This reciprocal translocation t(9;11)(p22;q23) resulted in the formation of MLL-AF9 fusion gene in the ALL patient [GenBank:AM050804]. Additionally, we identified a breakpoint which is identical with that identified in the ALL patient [GenBank:AM050804].

Conclusions

In summary, our current results reaffirm our previous findings that BA-induced apoptosis may cause chromosomal breakages in nasopharyngeal epithelial cells. In addition, our findings further implicate that MAR/SAR, which has a close association with topo II and CAD, plays a critical role in determining the positions of BA-induced chromosomal breakages. The positions of these BA-induced chromosome breaks shared high similarity with those identified in patients with leukaemia. Given that chromosomal breakage is an initial event of chromosome rearrangement and that cells may survive apoptosis upon compromised DNA repair, repeated exposure of nasopharyngeal epithelial cells to acid refluxate may contribute to genomic instability. The elevated mutation rate may in turn lead to the development of NPC. In order to clarify the relation between GOR and NPC, there are a few questions remain to be answered by further investigations: (i) Whether GOR may directly contribute to the pathogenesis of NPC through the cytotoxicity and genotoxicity driven by acid refluxate? (ii) Whether GOR may indirectly contribute to the pathogenesis of NPC through chronic inflammation of sinonasal tract (such as CRS) that has been recognised as a precursor of NPC? (iii) Whether both chronic inflammation of sinonasal tract and NPC share a similar underlying mechanism contributed by GOR? Nevertheless, our findings have unfolded the potential role of refluxed gastro-duodenal contents in contributing to NPC chromosome rearrangements.

Abbreviations

ABL :

Abelson murine leukaemia viral oncogene homolog 1

ALL:

Acute lymphoblastic leukaemia

AML:

Acute myelogenous leukaemia

BA:

Bile acid

CAD:

Caspase-activated DNase

CD:

Cluster of differentiation

CRS:

Chronic rhinosinusitis

EBV:

Epstein-Barr virus

EMT:

Epithelial-mesenchymal transition

EOR:

Extraoesophageal reflux

GOR:

Gastro-oesophageal reflux

GORD:

Gastro-oesophageal reflux disease

H2O2 :

Hydrogen peroxide

HMW:

High-molecular-weight

IFN:

Interferon

IL:

Interleukin

IPCR:

Inverse-PCR

MAR/SAR:

Matrix association region/scaffold attachment region

MDS:

Myelodysplastic syndrome

MLL :

Mixed lineage leukaemia

MMP:

Mitochondrial membrane potential

MRS:

MAR/SAR recognition signature

NF-kappa B:

Nuclear factor-kappa B

NPC:

Nasopharyngeal carcinoma

ORI:

Origin of replication

PS:

Phosphatidylserine

ROS:

Reactive oxygen species

SD:

Standard deviation

SDS:

Sodium dodecyl sulphate

t-AML:

Therapy-related AML

TNF-alpha:

Tumour necrosis factor-alpha

topo II:

Topoisomerase II

VP16:

Etoposide

References

  1. 1.

    Lo KW, Teo PM, Hui AB, To KF, Tsang YS, Chan SY, et al. High resolution allelotype of microdissected primary nasopharyngeal carcinoma. Cancer Res. 2000;60:3348–53.

    CAS  PubMed  Google Scholar 

  2. 2.

    Luo Z, Zhang L, Li Z, Li X, Li G, Yu H, et al. An in silico analysis of dynamic changes in microRNA expression profiles in stepwise development of nasopharyngeal carcinoma. BMC Med Genet. 2012;5:3.

    CAS  Google Scholar 

  3. 3.

    Sriamporn S, Vatanasapt V, Pisani P, Yongchaiyudha S, Rungpitarangsri V. Environmental risk factors for nasopharyngeal carcinoma: a case-control study in northeastern Thailand. Cancer Epidemiol Biomark Prev. 1992;1:345–8.

    CAS  Google Scholar 

  4. 4.

    Henle G, Henle W. Epstein-Barr virus-specific IgA serum antibodies as an outstanding feature of nasopharyngeal carcinoma. Int J Cancer. 1976;17:1–7.

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Raab-Traub N. Epstein-Barr virus and nasopharyngeal carcinoma. Semin Cancer Biol. 1992;3:297–307.

    CAS  PubMed  Google Scholar 

  6. 6.

    Armstrong RW, Armstrong MJ, Yu MC, Henderson BE. Salted fish and inhalants as risk factors for nasopharyngeal carcinoma in Malaysian Chinese. Cancer Res. 1983;43:2967–70.

    CAS  PubMed  Google Scholar 

  7. 7.

    Armstrong RW, Imrey PB, Lye MS, Armstrong MJ, Yu MC, Sani S. Nasopharyngeal carcinoma in Malaysian Chinese: occupational exposures to particles, formaldehyde and heat. Int J Epidemiol. 2000;29:991–8.

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Vaughan TL, Stewart PA, Teschke K, Lynch CF, Swanson GM, Lyon JL, et al. Occupational exposure to formaldehyde and wood dust and nasopharyngeal carcinoma. Occup Environ Med. 2000;57:376–84.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Henderson BE, Louie E, SooHoo JJ, Buell P, Gardner MB. Risk factors associated with nasopharyngeal carcinoma. N Engl J Med. 1976;295:1101–6.

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Yu MC, Garabrant DH, Huang TB, Henderson BE. Occupational and other non-dietary risk factors for nasopharyngeal carcinoma in Guangzhou, China. Int J Cancer. 1990;45:1033–9.

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Yuan JM, Wang XL, Xiang YB, Gao YT, Ross RK, Yu MC. Non-dietary risk factors for nasopharyngeal carcinoma in Shanghai, China. Int J Cancer. 2000;85:364–9.

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Ekburanawat W, Ekpanyaskul C, Brennan P, Kanka C, Tepsuwan K, Temiyastith S, et al. Evaluation of non-viral risk factors for nasopharyngeal carcinoma in Thailand: results from a case-control study. Asian Pac J Cancer Prev. 2010;11:929–32.

    PubMed  Google Scholar 

  13. 13.

    Huang WY, Lin CC, Jen YM, Lin KT, Yang MH, Chen CM, et al. Association between adult otitis media and nasopharyngeal cancer: a nationwide population-based cohort study. Radiother Oncol. 2012;104:338–42.

    PubMed  Article  Google Scholar 

  14. 14.

    Hung SH, Chen PY, Lin HC, Ting J, Chung SD. Association of rhinosinusitis with nasopharyngeal carcinoma: a population-based study. Laryngoscope. 2014;124:1515–20.

    PubMed  Article  Google Scholar 

  15. 15.

    Tsou YA, Lin CC, Tai CJ, Tsai MH, Tsai TC, Chen CM. Chronic rhinosinusitis and the risk of nasopharyngeal cancer in a Taiwanese health study. Am J Rhinol Allergy. 2014;28:168–72.

    PubMed  Article  Google Scholar 

  16. 16.

    Issing WJ, Karkos PD. Atypical manifestations of gastro-oesophageal reflux. J R Soc Med. 2003;96:477–80.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Fass R, Achem SR, Harding S, Mittal RK, Quigley E. Review article: supra-oesophageal manifestations of gastro-oesophageal reflux disease and the role of night-time gastro-oesophageal reflux. Aliment Pharmacol Ther. 2004;20(Suppl 9):26–38.

    PubMed  Article  Google Scholar 

  18. 18.

    Poelmans J, Tack J. Extraoesophageal manifestations of gastro-oesophageal reflux. Gut. 2005;54:1492–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Farrokhi F, Vaezi MF. Extra-esophageal manifestations of gastroesophageal reflux. Oral Dis. 2007;13:349–59.

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Madanick RD. Extraesophageal presentations of GERD: where is the science? Gastroenterol Clin N Am. 2014;43:105–20.

    Article  Google Scholar 

  21. 21.

    Dagli E, Yuksel A, Kaya M, Ugur KS, Turkay FC. Association of Oral Antireflux Medication with Laryngopharyngeal Reflux and Nasal Resistance. JAMA Otolaryngol Head Neck Surg. 2017;143:478–83.

    PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Gray ST, Sedaghat AR. Treatment of laryngopharyngeal reflux may decrease subjective symptoms of nasal congestion and objective measures of nasal resistance. JAMA Otolaryngol Head Neck Surg. 2017;143:483–4.

    PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Mosca F, Rossillo V, Leone CA. Manifestations of gastro-pharyngo-laryngeal reflux disease. Acta Otorhinolaryngol Ital. 2006;26:247–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Fink SM, Barwick KW, DeLuca V, Sanders FJ, Kandathil M, McCallum RW. The association of histologic gastritis with gastroesophageal reflux and delayed gastric emptying. J Clin Gastroenterol. 1984;6:301–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Bowrey DJ, Clark GW, Williams GT. Patterns of gastritis in patients with gastro-oesophageal reflux disease. Gut. 1999;45:798–803.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Little AG, DeMeester TR, Kirchner PT, O'Sullivan GC, Skinner DB. Pathogenesis of esophagitis in patients with gastroesophageal reflux. Surgery. 1980;88:101–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Dodds WJ, Dent J, Hogan WJ, Helm JF, Hauser R, Patel GK, et al. Mechanisms of gastroesophageal reflux in patients with reflux esophagitis. N Engl J Med. 1982;307:1547–52.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Shub MD, Ulshen MH, Hargrove CB, Siegal GP, Groben PA, Askin FB. Esophagitis: a frequent consequence of gastroesophageal reflux in infancy. J Pediatr. 1985;107:881–4.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Delahunty JE. Acid laryngitis. J Laryngol Otol. 1972;86:335–42.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Ward PH, Berci G. Observations on the pathogenesis of chronic non-specific pharyngitis and laryngitis. Laryngoscope. 1982;92:1377–82.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  31. 31.

    Ulualp SO, Toohill RJ, Hoffmann R, Shaker R. Pharyngeal pH monitoring in patients with posterior laryngitis. Otolaryngol Head Neck Surg. 1999;120:672–7.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32.

    Cherry J, Siegel CI, Margulies SI, Donner M. Pharyngeal localization of symptoms of gastroesophageal reflux. Ann Otol Rhinol Laryngol. 1970;79:912–4.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33.

    Barbero GJ. Gastroesophageal reflux and upper airway disease. Otolaryngol Clin N Am. 1996;29:27–38.

    CAS  Google Scholar 

  34. 34.

    Wise SK, Wise JC, DelGaudio JM. Association of nasopharyngeal and laryngopharyngeal reflux with postnasal drip symptomatology in patients with and without rhinosinusitis. Am J Rhinol. 2006;20:283–9.

    PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Poelmans J, Tack J, Feenstra L. Chronic middle ear disease and gastroesophageal reflux disease: a causal relation? Otol Neurotol. 2001;22:447–50.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. 36.

    Tasker A, Dettmar PW, Panetti M, Koufman JA, Birchall P, Pearson JP. Is gastric reflux a cause of otitis media with effusion in children? Laryngoscope. 2002;112:1930–4.

    PubMed  Article  PubMed Central  Google Scholar 

  37. 37.

    Serra A, Cocuzza S, Poli G, La Mantia I, Messina A, Pavone P. Otologic findings in children with gastroesophageal reflux. Int J Pediatr Otorhinolaryngol. 2007;71:1693–7.

    PubMed  Article  PubMed Central  Google Scholar 

  38. 38.

    Al Saab F, Manoukian JJ, Al Sabah B, Almot S, Nguyen LH, Tewfik TL, et al. Linking laryngopharyngeal reflux to otitis media with effusion: pepsinogen study of adenoid tissue and middle ear fluid. J Otolaryngol Head Neck Surg. 2008;37:565–71.

    Google Scholar 

  39. 39.

    Sontag SJ, O'Connell S, Khandelwal S, Miller T, Nemchausky B, Schnell TG, et al. Most asthmatics have gastroesophageal reflux with or without bronchodilator therapy. Gastroenterology. 1990;99:613–20.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Schnatz PF, Castell JA, Castell DO. Pulmonary symptoms associated with gastroesophageal reflux: use of ambulatory pH monitoring to diagnose and to direct therapy. Am J Gastroenterol. 1996;91:1715–8.

    CAS  PubMed  Google Scholar 

  41. 41.

    Mujica VR, Rao SS. Recognizing atypical manifestations of GERD. Asthma, chest pain, and otolaryngologic disorders may be due to reflux. Postgrad Med. 1999;105:53–6.

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Ulualp SO, Toohill RJ, Hoffmann R, Shaker R. Possible relationship of gastroesophagopharyngeal acid reflux with pathogenesis of chronic sinusitis. Am J Rhinol. 1999;13:197–202.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43.

    DelGaudio JM. Direct nasopharyngeal reflux of gastric acid is a contributing factor in refractory chronic rhinosinusitis. Laryngoscope. 2005;115:946–57.

    PubMed  Article  PubMed Central  Google Scholar 

  44. 44.

    Lin YH, Chang TS, Yao YC, Li YC. Increased risk of chronic sinusitis in adults with Gastroesophgeal reflux disease: a Nationwide population-based cohort study. Medicine (Baltimore). 2015;94:e1642.

    Article  Google Scholar 

  45. 45.

    DiBaise JK, Huerter JV, Quigley EM. Sinusitis and gastroesophageal reflux disease. Ann Intern Med. 1998;129:1078.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. 46.

    Contencin P, Narcy P. Nasopharyngeal pH monitoring in infants and children with chronic rhinopharyngitis. Int J Pediatr Otorhinolaryngol. 1991;22:249–56.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Contencin P, Viala P, Narcy P. Variations of nasopharyngeal pH in nasopharyngitis in children. Presse Med. 1991;20:1595–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Phipps CD, Wood WE, Gibson WS, Cochran WJ. Gastroesophageal reflux contributing to chronic sinus disease in children: a prospective analysis. Arch Otolaryngol Head Neck Surg. 2000;126:831–6.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  49. 49.

    Aydin E, Tastan E, Aydogan F, Arslan N, Karaca G. Role of nasopharyngeal reflux in the etiology of otitis media with effusion. J Otolaryngol Head Neck Surg. 2011;40:499–503.

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Pincus RL, Kim HH, Silvers S, Gold S. A study of the link between gastric reflux and chronic sinusitis in adults. Ear Nose Throat J. 2006;85:174–8.

    PubMed  Google Scholar 

  51. 51.

    Hashimoto K. The effect of reflux of bile juice on the development of residual stomach cancer. Nihon Geka Gakkai Zasshi. 1987;88:26–34.

    CAS  PubMed  Google Scholar 

  52. 52.

    Buchmann F, Baumer F, Eilles C. Significance of duodenogastric reflux in the pathogenesis of primary stomach cancer. Zentralbl Chir. 1988;113:170–3.

    CAS  PubMed  Google Scholar 

  53. 53.

    Lagergren J, Bergstrom R, Lindgren A, Nyren O. Symptomatic gastroesophageal reflux as a risk factor for esophageal adenocarcinoma. N Engl J Med. 1999;340:825–31.

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Shaheen N, Ransohoff DF. Gastroesophageal reflux, Barrett esophagus, and esophageal cancer: scientific review. JAMA. 2002;287:1972–81.

    PubMed  Article  Google Scholar 

  55. 55.

    Qadeer MA, Colabianchi N, Vaezi MF. Is GERD a risk factor for laryngeal cancer? Laryngoscope. 2005;115:486–91.

    PubMed  Article  Google Scholar 

  56. 56.

    Ward PH, Hanson DG. Reflux as an etiological factor of carcinoma of the laryngopharynx. Laryngoscope. 1988;98:1195–9.

    CAS  PubMed  Google Scholar 

  57. 57.

    Vereczkei A, Horvath OP, Varga G, Molnar TF. Gastroesophageal reflux disease and non-small cell lung cancer. Results of a pilot study. Dis Esophagus. 2008;21:457–60.

    CAS  PubMed  Article  Google Scholar 

  58. 58.

    Bernstein H, Bernstein C, Payne CM, Dvorakova K, Garewal H. Bile acids as carcinogens in human gastrointestinal cancers. Mutat Res. 2005;589:47–65.

    CAS  PubMed  Article  Google Scholar 

  59. 59.

    Sereg-Bahar M, Jerin A, Jansa R, Stabuc B, Hocevar-Boltezar I. Pepsin and bile acids in saliva in patients with laryngopharyngeal reflux - a prospective comparative study. Clin Otolaryngol. 2015;40:234–9.

    CAS  PubMed  Article  Google Scholar 

  60. 60.

    Sereg-Bahar M, Jerin A, Hocevar-Boltezar I. Higher levels of total pepsin and bile acids in the saliva as a possible risk factor for early laryngeal cancer. Radiol Oncol. 2015;49:59–64.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  61. 61.

    Shellman Z, Aldhahrani A, Verdon B, Mather M, Paleri V, Wilson J, et al. Bile acids: a potential role in the pathogenesis of pharyngeal malignancy. Clin Otolaryngol. 2017;42:969–73.

    CAS  PubMed  Article  Google Scholar 

  62. 62.

    Heerboth S, Housman G, Leary M, Longacre M, Byler S, Lapinska K, et al. EMT and tumor metastasis. Clin Transl Med. 2015;4:6.

    PubMed  PubMed Central  Article  Google Scholar 

  63. 63.

    Goldstein MG, Li Z. Heat-shock proteins in infection-mediated inflammation-induced tumorigenesis. J Hematol Oncol. 2009;2:5.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  64. 64.

    Li X, Fang P, Mai J, Choi ET, Wang H, Yang XF. Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases and cancers. J Hematol Oncol. 2013;6:19.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. 65.

    Wang WT, Ye H, Wei PP, Han BW, He B, Chen ZH, et al. LncRNAs H19 and HULC, activated by oxidative stress, promote cell migration and invasion in cholangiocarcinoma through a ceRNA manner. J Hematol Oncol. 2016;9:117.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. 66.

    Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med. 2010;49:1603–16.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  67. 67.

    Perkins ND. The diverse and complex roles of NF-kappaB subunits in cancer. Nat Rev Cancer. 2012;12:121–32.

    CAS  PubMed  Article  Google Scholar 

  68. 68.

    Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer. 2007;121:2381–6.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  69. 69.

    Sasaki CT, Issaeva N, Vageli DP. In vitro model for gastroduodenal reflux-induced nuclear factor-kappaB activation and its role in hypopharyngeal carcinogenesis. Head Neck. 2016;38:E1381–91.

    PubMed  Article  Google Scholar 

  70. 70.

    Lawrence T. The nuclear factor NF−+¦B pathway in inflammation. Cold Spring Harb Perspect Biol. 2009;1:a001651.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  71. 71.

    Dokka S, Shi X, Leonard S, Wang L, Castranova V, Rojanasakul Y. Interleukin-10-mediated inhibition of free radical generation in macrophages. Am J Physiol Lung Cell Mol Physiol. 2001;280:L1196.

    CAS  PubMed  Article  Google Scholar 

  72. 72.

    Wang CS, Liu SH, Peng J, Tang C, Zhu WG. Bile acids cycle disruption in patients with nasopharyngeal carcinoma promotes the elevation of interleukin-10 secretion. Afr Health Sci. 2015;15:1200–3.

    PubMed  PubMed Central  Article  Google Scholar 

  73. 73.

    Latorre E, Matheus N, Layunta E, Alcalde AI, Mesonero JE. IL-10 counteracts proinflammatory mediator evoked oxidative stress in caco-2 cells. Mediat Inflamm. 2014;2014:1–6.

    Article  CAS  Google Scholar 

  74. 74.

    Moreira AP, Dias-Melicio LA, Soares AMVC. Interleukin-10 but not transforming growth factor beta inhibits murine activated macrophages Paracoccidioides brasiliensis killing: effect on H2O2 and NO production. Cell Immunol. 2010;263:196–203.

    CAS  PubMed  Article  Google Scholar 

  75. 75.

    Tan SN, Sim SP. Bile acids at neutral and acidic pH induce apoptosis and gene cleavages in nasopharyngeal epithelial cells: implications in chromosome rearrangement. BMC Cancer. 2018;18:409.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  76. 76.

    Bernstein C, Bernstein H, Garewal H, Dinning P, Jabi R, Sampliner RE, et al. A bile acid-induced apoptosis assay for colon cancer risk and associated quality control studies. Cancer Res. 1999;59:2353–7.

    CAS  PubMed  Google Scholar 

  77. 77.

    Bernstein C, Payne CM, Bernstein H. Bile Acids: Promoters or Carcinogens in Colon Cancer? J Carcinogene Mutagene. 2011;2:101e.

    Article  Google Scholar 

  78. 78.

    Fang Y, Chen X, Bajpai M, Verma A, Das KM, Souza RF, et al. Cellular origins and molecular mechanisms of Barrett's esophagus and esophageal adenocarcinoma. Ann N Y Acad Sci. 2013;1300:187–99.

    CAS  PubMed  Article  Google Scholar 

  79. 79.

    Filipski J, Leblanc J, Youdale T, Sikorska M, Walker PR. Periodicity of DNA folding in higher order chromatin structures. EMBO J. 1990;9:1319–27.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  80. 80.

    Brown DG, Sun XM, Cohen GM. Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J Biol Chem. 1993;268:3037–9.

    CAS  PubMed  Google Scholar 

  81. 81.

    Oberhammer F, Wilson JW, Dive C, Morris ID, Hickman JA, Wakeling AE, et al. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 1993;12:3679–84.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  82. 82.

    Compton MM. A biochemical hallmark of apoptosis: internucleosomal degradation of the genome. Cancer Metastasis Rev. 1992;11:105–19.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  83. 83.

    Bortner CD, Oldenburg NB, Cidlowski JA. The role of DNA fragmentation in apoptosis. Trends Cell Biol. 1995;5:21–6.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  84. 84.

    Lechardeur D, Xu M, Lukacs GL. Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells. J Cell Biol. 2004;167:851–62.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  85. 85.

    Cockerill PN, Garrard WT. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell. 1986;44:273–82.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  86. 86.

    Shao JY, Wang HY, Huang XM, Feng QS, Huang P, Feng BJ, et al. Genome-wide allelotype analysis of sporadic primary nasopharyngeal carcinoma from southern China. Int J Oncol. 2000;17:1267–75.

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    van Drunen CM, Sewalt RG, Oosterling RW, Weisbeek PJ, Smeekens SC, van Driel R. A bipartite sequence element associated with matrix/scaffold attachment regions. Nucleic Acids Res. 1999;27:2924–30.

    PubMed  PubMed Central  Article  Google Scholar 

  88. 88.

    Strissel PL, Strick R, Tomek RJ, Roe BA, Rowley JD, Zeleznik L. DNA structural properties of AF9 are similar to MLL and could act as recombination hot spots resulting in MLL/AF9 translocations and leukemogenesis. Hum Mol Genet. 2000;9:1671–9.

    CAS  PubMed  Article  Google Scholar 

  89. 89.

    Strick R, Zhang Y, Emmanuel N, Strissel PL. Common chromatin structures at breakpoint cluster regions may lead to chromosomal translocations found in chronic and acute leukemias. Hum Genet. 2006;119:479–95.

    CAS  PubMed  Article  Google Scholar 

  90. 90.

    Tan SN, Sim SP, Khoo AS. Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma. Hum Genomics. 2018;12:29.

    PubMed  PubMed Central  Article  Google Scholar 

  91. 91.

    Frisch M, Frech K, Klingenhoff A, Cartharius K, Liebich I, Werner T. In silico prediction of scaffold/matrix attachment regions in large genomic sequences. Genome Res. 2002;12:349–54.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  92. 92.

    Singh GB, Kramer JA, Krawetz SA. Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucleic Acids Res. 1997;25:1419–25.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  93. 93.

    Dvorak K, Payne CM, Chavarria M, Ramsey L, Dvorakova B, Bernstein H, et al. Bile acids in combination with low pH induce oxidative stress and oxidative DNA damage: relevance to the pathogenesis of Barrett's oesophagus. Gut. 2007;56:763–71.

    CAS  PubMed  Article  Google Scholar 

  94. 94.

    Nehra D, Howell P, Williams CP, Pye JK, Beynon J. Toxic bile acids in gastro-oesophageal reflux disease: influence of gastric acidity. Gut. 1999;44:598–602.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  95. 95.

    Iftikhar SY, Ledingham S, Steele RJ, Evans DF, Lendrum K, Atkinson M, et al. Bile reflux in columnar-lined Barrett's oesophagus. Ann R Coll Surg Engl. 1993;75:411–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  96. 96.

    Junqueira JC, Penna FJ. Nasopharyngeal pH and gastroesophageal reflux in children with chronic respiratory disease. J Pediatr. 2007;83:225–32.

    Google Scholar 

  97. 97.

    Tan SN, Sim SP, Khoo AS. Potential role of oxidative stress-induced apoptosis in mediating chromosomal rearrangements in nasopharyngeal carcinoma. Cell Biosci. 2016;6:35.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  98. 98.

    Ebert SN, Shtrom SS, Muller MT. Topoisomerase II cleavage of herpes simplex virus type I DNA in vivo is replication dependent. J Virol. 1990;64:4059–66.

    CAS  PubMed  PubMed Central  Google Scholar 

  99. 99.

    Broeker PL, Super HG, Thirman MJ, Pomykala H, Yonebayashi Y, Tanabe S, et al. Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia: correlation with scaffold attachment regions and topoisomerase II consensus binding sites. Blood. 1996;87:1912–22.

    CAS  PubMed  Google Scholar 

  100. 100.

    Jeffs AR, Benjes SM, Smith TL, Sowerby SJ, Morris CM. The BCR gene recombines preferentially with Alu elements in complex BCR-ABL translocations of chronic myeloid leukaemia. Hum Mol Genet. 1998;7:767–76.

    CAS  PubMed  Article  Google Scholar 

  101. 101.

    Cynthia PN, Sim SP. Etoposide-induced apoptosis results in chromosome breaks within the AF9 gene: its implication in chromosome rearrangement in leukaemia. Adv Biosci Biotechnol. 2012;3:686–94.

    Article  CAS  Google Scholar 

  102. 102.

    Bohnhorst I, Jawad S, Lange B, Kjeldsen J, Hansen JM, Kjeldsen AD. Prevalence of chronic rhinosinusitis in a population of patients with gastroesophageal reflux disease. Am J Rhinol Allergy. 2015;29:e70–4.

    PubMed  Article  Google Scholar 

  103. 103.

    Chodosh PL. Gastro-esophago-pharyngeal reflux. Laryngoscope. 1977;87:1418–27.

    CAS  PubMed  Article  Google Scholar 

  104. 104.

    Contencin P, Maurage C, Ployet MJ, Seid AB, Sinaasappel M. Gastroesophageal reflux and ENT disorders in childhood. Int J Pediatr Otorhinolaryngol. 1995;32(Suppl):S135–44.

    PubMed  Article  PubMed Central  Google Scholar 

  105. 105.

    Jaspersen D. Extra-esophageal disorders in gastroesophageal reflux disease. Dig Dis. 2004;22:115–9.

    PubMed  Article  PubMed Central  Google Scholar 

  106. 106.

    Blumin JH, Merati AL, Toohill RJ. Duodenogastroesophageal reflux and its effect on extraesophageal tissues: a review. Ear Nose Throat J. 2008;87:234–7.

    PubMed  Google Scholar 

  107. 107.

    Aldhahrani A, Verdon B, Ward C, Pearson J. Effects of bile acids on human airway epithelial cells: implications for aerodigestive diseases. ERJ Open Res. 2017;3.

  108. 108.

    Herbella FA, Neto SP, Santoro IL, Figueiredo LC. Gastroesophageal reflux disease and non-esophageal cancer. World J Gastroenterol. 2015;21:815–9.

    PubMed  PubMed Central  Article  Google Scholar 

  109. 109.

    Sperry AO, Blasquez VC, Garrard WT. Dysfunction of chromosomal loop attachment sites: illegitimate recombination linked to matrix association regions and topoisomerase II. Proc Natl Acad Sci U S A. 1989;86:00107-16.

  110. 110.

    Blasquez VC, Sperry AO, Cockerill PN, Garrard WT. Protein:DNA interactions at chromosomal loop attachment sites. Genome. 1989;31:503–9.

    CAS  PubMed  Article  Google Scholar 

  111. 111.

    Razin SV, Shen K, Ioudinkova E, Scherrer K. Functional analysis of DNA sequences located within a cluster of DNase I hypersensitive sites colocalizing with a MAR element at the upstream border of the chicken alpha-globin gene domain. J Cell Biochem. 1999;74:38–49.

    CAS  PubMed  Article  Google Scholar 

  112. 112.

    Swansbury GJ, Slater R, Bain BJ, Moorman AV, Secker-Walker LM. Hematological malignancies with t(9;11)(p21-22;q23)--a laboratory and clinical study of 125 cases. European 11q23 workshop participants. Leukemia. 1998;12:792–800.

    CAS  PubMed  Article  Google Scholar 

  113. 113.

    Donev R, Horton R, Beck S, Doneva T, Vatcheva R, Bowen WR, et al. Recruitment of heterogeneous nuclear ribonucleoprotein A1 in vivo to the LMP/TAP region of the major histocompatibility complex. J Biol Chem. 2003;278:5214–26.

    CAS  PubMed  Article  Google Scholar 

  114. 114.

    Ostermeier GC, Liu Z, Martins RP, Bharadwaj RR, Ellis J, Draghici S, et al. Nuclear matrix association of the human b-globin locus utilizing a novel approach to quantitative real-time PCR. Nucleic Acids Res. 2003;31:3257–66.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  115. 115.

    Thilmony R, Guttman ME, Lin JW, Blechl AE. The wheat HMW-glutenin 1Dy10 gene promoter controls endosperm expression in Brachypodium distachyon. GM Crops Food. 2014;5:36–43.

    PubMed  Article  Google Scholar 

  116. 116.

    Kas E, Chasin LA. Anchorage of the Chinese hamster dihydrofolate reductase gene to the nuclear scaffold occurs in an intragenic region. J Mol Biol. 1987;198:677–92.

    CAS  PubMed  Article  Google Scholar 

  117. 117.

    Romig H, Ruff J, Fackelmayer FO, Patil MS, Richter A. Characterisation of two intronic nuclear-matrix-attachment regions in the human DNA topoisomerase I gene. Eur J Biochem. 1994;221:411–9.

    CAS  PubMed  Article  Google Scholar 

  118. 118.

    Mielke C, Kohwi Y, Kohwi-Shigematsu T, Bode J. Hierarchical binding of DNA fragments derived from scaffold-attached regions: correlation of properties in vitro and function in vivo. Biochemistry. 1990;29:7475–85.

    CAS  PubMed  Article  Google Scholar 

  119. 119.

    Bode J, Kohwi Y, Dickinson L, Joh T, Klehr D, Mielke C, et al. Biological significance of unwinding capability of nuclear matrix-associating DNAs. Science. 1992;255:195–7.

    CAS  PubMed  Article  Google Scholar 

  120. 120.

    Bode J, Schlake T, Rios-Ramirez M, Mielke M, Stengert M, Kay V, et al. Scaffold/matrix-attached regions: structural properties creating transcriptionally active loci. Int Rev Cytol. 1995;162A:389–454.

    CAS  PubMed  PubMed Central  Google Scholar 

  121. 121.

    Boulikas T. Chromatin domains and prediction of MAR sequences. Int Rev Cytol. 1995;162A:279–388.

    CAS  PubMed  Google Scholar 

  122. 122.

    Mimori K, Druck T, Inoue H, Alder H, Berk L, Mori M, et al. Cancer-specific chromosome alterations in the constitutive fragile region FRA3B. Proc Natl Acad Sci U S A. 1999;96:7456–61.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  123. 123.

    Arlt MF, Miller DE, Beer DG, Glover TW. Molecular characterization of FRAXB and comparative common fragile site instability in cancer cells. Genes Chromosomes Cancer. 2002;33:82–92.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  124. 124.

    Bae YS, Kawasaki I, Ikeda H, Liu LF. Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro. Proc Nati Acad Sci USA. 1988;85:2076–80.

    CAS  Article  Google Scholar 

  125. 125.

    Gale KC, Osheroff N. Intrinsic intermolecular DNA Ligation activity of Eukaryotic Topoisomerase II. Potential roles in recombination. J Biol Chem. 1992;267:12090–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  126. 126.

    Kas E, Poljak L, Adachi Y, Laemmli UK. A model for chromatin opening: stimulation of topoisomerase II and restriction enzyme cleavage of chromatin by distamycin. EMBO J. 1993;12:115–26.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  127. 127.

    Xiao H, Li TK, Yang JM, Liu LF. Acidic pH induces topoisomerase II-mediated DNA damage. Proc Natl Acad Sci U S A. 2003;100:5205–10.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  128. 128.

    Li TK, Chen AY, Yu C, Mao Y, Wang H, Liu LF. Activation of topoisomerase II-mediated excision of chromosomal DNA loops during oxidative stress. Genes Dev. 1999;13:1553–60.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We sincerely thank Prof. Tsao Sai Wah (The University of Hong Kong, Hong Kong, China) and Prof. Lo Kwok Wai (The Chinese University of Hong Kong, Hong Kong, China) for providing the normal nasopharyngeal epithelial cell line (NP69). We express our gratitude to Prof. Sam Choon Kook (formerly from University of Malaya, Malaysia) for providing the NPC cell line (TWO4). We are deeply grateful to Universiti Malaysia Sarawak for all infrastructure support. We would like to acknowledge Ministry of Higher Education Malaysia for the MyBrain15 (MyPhd) Postgraduate Scholarship and the Fundamental Research Grant [FRGS/SKK01(01)/977/2013 (18)] that supported this work.

Funding

The project was funded by Ministry of Higher Education Malaysia [MOHE Fundamental Research Grant, grant number: FRGS/SKK01(01)/977/2013 (18)].

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Author information

Affiliations

  1. Faculty of Medicine and Health Sciences, Department of Paraclinical Sciences, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia
    • Sang-Nee Tan
    •  & Sai-Peng Sim
Authors
  1. Sang-Nee Tan
    View author publications
    You can also search for this author in
  2. Sai-Peng Sim
    View author publications
    You can also search for this author in

Contributions

SNT conducted all the experimental work, carried out the in silico predictions of chromatin structural elements (MAR/SAR, repeat element and topo II cleavage site), performed data analyses, constructed the genomic maps and wrote the manuscript. SPS contributed to the conception and design of the study, interpreted the data, edited the manuscript and supervised the whole project. Both authors read and approved the final manuscript.

Corresponding author

Correspondence to Sai-Peng Sim.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Additional files

Additional file 1:

Flow chart depicting the simplified DNA manipulation steps in preparation for nested IPCR. (PDF 46 kb)

Additional file 2:

Description of exons and introns in the AF9 gene. (PDF 64 kb)

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tan, S., Sim, S. Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells. BMC Med Genomics 12, 9 (2019). https://doi.org/10.1186/s12920-018-0465-4

Download citation

Keywords

  • Chronic rhinosinusitis
  • Nasopharyngeal carcinoma
  • Gastro-oesophageal reflux
  • Bile acid
  • Apoptosis
  • AF9
  • Matrix association region/scaffold attachment region

BMC Medical Genomics

ISSN: 1755-8794

Contact us