Comprehensive analysis of key genes and microRNAs in radioresistant nasopharyngeal carcinoma

Radiotherapy is a mainly treatment for nasopharyngeal carcinoma (NPC). However, radioresistance is one of the major factors to affect the therapeutic efficacy and prognosis of patients [1–3]. Accordingly, identifying potential biomarkers and studying the molecular mechanisms associated with radioresistant nasopharyngeal carcinoma has become a hot topic both in basic and clinical research.

Microarrays are considered to be an important method for identifying potential biomarkers in many diseases at the molecular level with more effective and detailed insights [4]. Several microRNAs and mRNAs have been discovered to be involved in radioresistant NPC, whereas traditional methods have failed to elucidate the interaction of mRNAs and microRNAs and the molecular mechanisms of NPC due to the limitations on the comparative analysis [5–7]. Therefore, systematically investigating the interaction between microRNA and mRNA, and elucidating the molecular mechanism of radioresistant NPC is of great significance. With the development of bioinformatics, we can apply global analysis to process the data generated by microarray technology and find the interaction between DEGs and DEM, especially in the pathway interaction network, to summarize their potential mechanisms in diseases [8–10]. Based on above mentioned reasons, the present study aims to identify the key genes and miRNAs and to explore their potential molecular mechanisms in radioresistant nasopharyngeal carcinoma.

In this study, we analysis the differentially expressed genes and microRNA between radioresistant NPC CNE2-R cells and radiosensitive CNE2 cells based on the database of GSE48501 and GSE48502, and then employed bioinformatics to analyze the pathways and GO terms in which DEGs and DEMS target genes are involved. Moreover, Construction of protein-protein interaction network and identification of hub genes. Finally, analyzed the biological networks for validated target gene of hub miRNAs. Our data may provide an important contribution to identified biological markers and clarify the mechanisms of NPC radioresistance.

Nasopharyngeal carcinoma is a geographically distributed disease, especially in southern china and southeast Asia. Although radiotherapy is considered to be the primary treatment for NPC, radioresistance-induced locoregional recurrence and distant metastases remains mainly obstacle to successful treatment [11, 12]. Therefore, in-depth study biomarkers and mechanisms of radioresistance in NPC is of great significance for improving the radiosensitivity of NPC and provide a new ideas for design of good therapy.

Previous studies have identified that radioresistance-associated molecules (mRNAs, microRNAs, and proteins) regulate radioresistance through different biological process, such as DNA repair, apoptosis, cell cycle, and protective autophagy [9]. However, the molecular mechanisms underlying NPC radioresistnce remain elusive.

In order to understand the mechanisms underlying in radioresistant NPC, we performed global analysis of key genes and microRNAs in radioresistant Nasopharyngeal Carcinoma by bioinformatics analysis. Our result demonstrated that a total of 373 DEGs and 14 DEMs between radioresistant NPC CNE2-IR cells and radiosensitive CNE2 cells were identified. Our study revealed that JUN was significantly up-regulated in radioresistant NPC CNE2-IR cell and which was overlap between the DEGs and the DigSee. Meanwhile, in Protein-Protein interaction network analysis, JUN was a core hub gene in PPI network. Recent research has shown that JUN is an important components of the activator protein-1 (AP-1) transcription factor and is closely related to cell proliferation, apoptosis and malignant transformation [13]. JUN could promote tumor growth and progression. Over-expression of c-jun was found to result in abnormal cell proliferation and loss of apoptosis. Some researchers have noted that inhibition the expression of c-jun can enhances radiosensitivity, induces cell cycle arrest and apoptosis [14]. Our previous study shown that the expression of c-jun was significantly up-regulated in CNE-2R cells, which may be associated with the radioresistance of NPC [15]. The kegg pathway analysis data indicated that JUN is involved in a variety of pathways. Such as TNF signaling pathway, Epstein-Barr virus infection, MAPK signaling pathway, Pathways in cancer. MAPK signaling activity confers inherent radioresistance to KRAS-mutant colorectal carcinoma cells by rapidly upregulating of heterogeneous nuclear ribonucleoprotein K (hnRNPK) [16]. Our result showed that JUN was involved in Hepatitis B, NOD-like receptor signaling pathway, AGE-RAGE signaling pathway in diabetic complications, Pathways in cancer, IL-17 signaling pathway, TNF signaling pathway (Fig. 3b). We speculated that JUN may be participated in the regulation of nasopharyngeal carcinoma radiosensitivity through these pathways, the internal mechanism may need to be further tested in the future experiments. Based on the above, we conclude that JUN is involved in NPC radioresistance, which may provide new clues to improve radiosensitivity of Nasopharyngeal Carcinoma.

microRNAs are single stranded, endogenous, 19–25 nucleotide (nt), which are thought to be modulated tumor radiosensitivity [17]. Therefore, identification of radioresistance associated miRNAs which may contribute to more effective treatments for NPC patients. In this study, we screened out 14 differentially expressed miRNAs in the radioresistant CNE2-R cells, including the up-regulated mirRNA-762, mirRNA-1202, mirRNA-193b, mirRNA-let-7e and down-regulated mirRNA-203, mirRNA-545, mirRNA-4291, mirRNA-183, mirRNA-24, mirRNA-130a, miRNA-660, miRNA-31, miRNA-30a, miRNA-23a, suggesting that the regulation of these miRNAs might be participate in the NPC radioresistance. Most of them have been shown to be associated with radioresistance of tumor [2, 18, 19]. mirRNA-let-7e and miRNA-31 have been recently discovered to involving in the acquisition of cancer cell radioresistance [1, 19]. Recent reports indicate that miRNA-23a is downregulated in the radioresistant NPC tissues, and is an independent predictor of poor prognosis in patients with nasopharyngeal carcinoma. up-regulated miRNA-23a improves NPC cell radiosensitivity in vivo and vitro. Downregulated miRNA-23a increases NPC radioresistance through activating IL-8/Stat3 signaling. Targeting miR-23a/IL-8/Stat3 signaling might be an effective approach to improving radiosensitivity of NPC [18]. It has been reported that miR-24 is frequently downregulated in NPC cell lines, and the consumption of miR-24 inhibited NPC cell growth and proliferation, while improving the radiosensitivity of NPC both in vitro and in vivo. In addition, it is reported that SP1 was verified as a target for miR-24, miR-24/SP pathway should help us understand the radiosensitivity mechanisms of human NPC, which may be a potential therapeutic target [20]. Our study detected that miRNA-24 is down-regulated in radioresistant NPC cells, and which not only was common in DEMs and JUN-related microRNAs, but also had significantly enriched in P53 signaling pathway. In conclusion, we can infer that the above mention microRNAs, especially miRNA-24 may be a key factor to affect the radiosensitivity of NPC, which may be helpful to predict radiosensitivity in NPC.

To completely understand the function of miRNAs and mRNAs in radioresistant NPC, we performed pathway enrichment analysis of DEGs and DEMs, the result demonstrated that 4 pathways are considered as the key pathways for the radiosensitivity of NPC, including TGF-beta signaling pathway, Hepatitis B, Pathways in cancer and p53 signaling pathway, which were regulated by miRNA and mRNA together. Moreover, hsa-miR-24 and its target gene were found have significantly enriched in P53 signaling pathway. P53 can predict cancer response to IR and chemotherapy [21]. Improving the radiosensitivity of non-small cell lung cancer cells by inhibition of TGF-β1 signaling [22]. It is reported that p53 signaling pathway correlates with the radioresponse of non-small cell lung cancer. Differentially expressed genes in the p53 signaling pathway related to DNA damage repair, apoptosis, cycle regulation, metastasis, deterioration and radioresistance [23]. Previous study has shown that p53 signaling pathway mediate inhibition and apoptosis induced by 12C6+ heavy ion beam irradiation on HepG2 cancer cells [24]. Accordingly, we can infer that hsa-miR-24 and p53 signaling pathway should provide an important contribution to understand the mechanisms of radiosensitivity in human NPC and that it may represent a potential therapeutic target.

In conclusion, this study demonstrates that the upregulated gene JUN was overlap both in DEGs and publicly available studies, was a core hub gene in PPI network, which was potentially targeted by three miRNAs, including hsa-miR-203, hsa-miR-24 and hsa-miR-31. Pathway analysis showed that both up-regulated gene and DEMs were enriched in TGF-beta signaling pathway, Hepatitis B, Pathways in cancer and p53 signaling pathway. Finally, we analyzed the biological networks for validated target gene of common miRNAs, the result indicated that miR-24 is frequently down-regulated in radioresistant NPC cell lines and significantly enriched in P53 signaling pathway. Based on these reasons, our study indicated that the JUN, miR-24 and P53 signaling pathway may be associated with radioresistance in Nasopharyngeal Carcinoma, and which may provide new clues for improving radiosensitivity in Nasopharyngeal Carcinoma. However, these results are only speculated by the combination of databases and bioinformatics methods, and still needs to be confirmed. In the following study, we explored the association between JUN expression and radioresistance in vitro. To further determine the clinical predictive value of JUN in NPC, we performed immunohistochemistry assays to examine the protein expression pattern of JUN in NPC specimens and normal nasopharyngeal epithelium specimens. And then, we performed a log-rank test analysis the Overall survival (OS) of patients with NPC based on JUN expression. Finally, Study the correlation between JUN expression and distant metastasis in patients with NPC.