Mitochondrial proteomics of nasopharyngeal carcinoma metastasis
© Liu et al.; licensee BioMed Central Ltd. 2012
Received: 14 August 2012
Accepted: 30 November 2012
Published: 6 December 2012
Mitochondrial proteomic alterations of nasopharyngeal carcinoma metastasis remain unknown. Our purpose is to screen mitochondrial proteins for the elucidation of the molecular mechanisms of nasopharyngeal carcinoma metastasis and the discovery of metastasis-related biomarkers.
Mitochondria were isolated from nasopharyngeal carcinoma metastatic (5-8F) and nonmetastatic (6-10B) cell lines, respectively. After characterization of isolated mitochondria, mitochondrial differentially expressed proteins (DEPs) were quantified by two-dimensional difference in-gel electrophoresis (2D-DIGE), and identified by peptide mass fingerprint (PMF) and tandem mass spectrometry (MS/MS). A functional enrichment analysis and a protein-protein interaction sub-network analysis for DEPs were carried out with bioinformatics. Furthermore, siRNAs transient transfections were used to suppress expressions of some up-regulated DEPs in metastatic cells (5-8F), followed by Transwell Migration assay.
Sixteen mitochondrial DEPs including PRDX3 and SOD2 were identified. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. The functional enrichment analyses of DEPs discovered five significant biological processes including cellular response to reactive oxygen species, hydrogen peroxide metabolic process, regulation of mitochondrial membrane potential, cell redox homeostasis and oxidation reduction, and five significant molecular functions including oxidoreductase activity, caspase inhibitor activity, peroxiredoxin activity, porin activity and antioxidant activity. A protein-protein interaction sub-network of DEPs was generated with literature data. Ten mitochondrial DEPs including PRDX3, PRDX6, SOD2, ECH1, SERPINB5, COX5A, PDIA5, EIF5A, IDH3B, and PSMC4 were rationalized in the tumor-stroma co-evolution model that mitochondrial oxidative stress directly contributes to tumor metastasis.
Sixteen mitochondrial DEPs were identified with mass spectrometry and ten of them were rationalized in the tumor-stroma co-evolution model. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. These data suggest that those mitochondrial DEPs are potential biomarkers for NPC metastasis, and their dysregulation would play important roles in mitochondria oxidative stress-mediated NPC metastatic process.
KeywordsNasopharyngeal carcinoma Tumor metastasis Mitochondria Proteome Differentially expressed proteins Functional enrichment analysis Literature data mining PRDX3
Nasopharyngeal carcinoma (NPC) cell lines 5-8F and 6-10B are derived from the same human cell line SUNE1 that is derived from a female poorly differentiated nasopharyngeal squamous epithelial cell carcinoma. Those two cell sublines 5-8F and 6-10B with the same genetic background have tumorigenic ability that was confirmed by the colony formation in soft agar and in vivo mouse model, but are quite different in metastatic potential [1–3]. 5-8F cells are metastatic and 6-10B cells are nonmetastatic, which was confirmed by in vivo mouse model . This different biological character shows that these two cell sublines are the useful experiment materials to study the molecular events of NPC metastasis. Based on those two subcelines, some research groups analyzed the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) using the whole-cell samples of those two cell lines. A total of 78 DEGs were identified with subtractive suppression hybridization  and microarray . Of them, 11 DEGs including PTHLH formed a metastasis-related gene network . A total of 28 DEPs were identified with two-dimensional gel electrophoresis (2DGE) coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) [6, 7]. Those DEPs were related to apoptosis, cell cycle, cell signal transduction, cell survival, transcription regulation, cell mobility, protein synthesis, and DNA damage repair [6, 7]. However, those DEGs and DEPs were not fully rationalized in the biological process of NPC metastasis, and there is no any overlap between those 78 DEGs and 28 DEPs [4–7]. It would be derived from the following limitation factors: (i) the range of the protein abundance of the whole-cell sample is too wide, not all proteins were extracted; (ii) 2DGE only can array very small partial components of a whole-cell proteome, and (iii) the process of translation of DEG to DEP is regulated by multiple factors, which results in the low consistence between the DEG and DEP profiles. The subcellular proteomics such as just focusing on one compartment of a cell would more effectively discover function-related proteins because the subcellular proteome is much simpler than the whole-cell proteome, it can maximally identify the protein components in a subcellular proteome.
Many evidences have demonstrated that mitochondria is the central to oxidative stress, and tumor cell self has anti-oxidant ability for its survival and metastasis [8–15]. Mitochondria are not just innocent bystander in cancer, but are related to tumorigenesis, apoptosis, cancer therapy and metastasis. The contribution of mitochondria to cancer involves three critical processes including alterations in glucose metabolism, production of reactive oxygen species (ROS), and compromise of intrinsic apoptotic function [8, 9]. According to the tumor-stroma co-evolution model opinion, cancer cells secrete hydrogen peroxide, induce oxidative stress in adjacent stromal cells, and result in an excess production of ROS in mitochondria. Excess stromal ROS induces cytoskeleton rearrangements, autophagy, and mitophagy in the tumor microenvironment to help cancer cells escape excess ROS in the primary tumor site, also induces the stromal over-production of recycled nutrients such as ketones and L-lactate that are fuel for tumor growth and metastasis [10–14]. This model provides fresh insight of how mitochondria involved in tumor metastasis. Therefore, a tight relationship exists between mitochondria and tumor metastasis. However, the causes, consequences, and other series of related questions regarding molecular mechanism of mictochondria in tumor metastasis still remain unclear. Thus, this current work focused on the mitochondrial proteome difference between metastatic 5-8F and 6-10B cells, and provided clues to clarification of the functional role of mitochondria in the process of NPC metastasis.
In order to screen mitochondrial proteins for the elucidation of the mechanism of NPC metastasis and the discovery of metastasis-related biomarkers, two-dimensional difference in-gel electrophoresis (2D-DIGE) was used to determine mitochondrial DEPs between metastatic 5-8F and nonmetastastic 6-10B cells, followed by protein characterization with peptide mass fingerprint (PMF) and tandem mass spectrometry (MS/MS). siRNA transient transfection was used to suppress the expressions of up-regulated DEPs in metastatic 5-8F cells, followed by the observation of changes in cell mobility potential by Transwell Migration assay. Cytoscape BiNGO plugin was used for gene ontology (GO) analyses of DEPs to identify the enriched biological processes and molecular functions. A protein-protein interaction sub-network of DEPs was generated with literature data mining from HPRD and NCBI databases. Ten DEPs were rationalized in a tumor-stroma co-evolution model, and associated with tumor metastasis.
Characterization of isolated mitochondria
Detection of mitochondrial DEPs between metastatic and nonmetastatic cells by 2D-DIGE
Identification of mitochondrial DEPs between metastatic and nonmetastatic cells by PMF and MS/MS
Differentially expressed proteins in mitochondria of metastatic 5-8F compared to non-metastatic 6-10B cells, identified by 2D-DIGE and MS (Ratio = 5-8F/6-10B; +, up-regulated; -, down-regulated)
Sequence coverage (%)
1. Oxidoreductase Proteins
[Mn], mitochondrial isoform B precursor
[NAD] subunit alpha, mitochondrial precursor
Protein disulfide isomerase related protein 5
Cytochrome c oxidase subunit 5A, mitochondrial precursor
2. Porin Proteins
Volatage-dependent anion selective channel protein 1
Volatage-dependent anion channel 2
3. Metabolism and regulation Proteins
Delta(3.5)-Delta(2.4)-dienovl-CoA isomerase, mitochondrial precursor
Eukaryotic initiation factor 5A
26S protease regulatory subunit 7 isoform 1
Ribosomal protein L7/L12, mitochondria
Calumenin isoform c precursor
Functional enrichment analyses of mitochondrial DEPs and protein-protein interaction data
Enriched Go terms of differentially expressed proteins in mitochondria of metastatic 5-8F relative to non-metastatic 6-10B cells
Cellular response to reactive oxygen species
PRDX6↑, PRDX3↑, SOD2↑
Hydrogen peroxide metabolic process
PRDX6↑, PRDX3↑, SOD2↑
Regulation of mitochondria membrane potential
Cell redox homeostasis
PRDX6↑, PDIA5↑, PRDX3↑
PRDX6↑, IDH3B↓, PDIA5↑, PRDX3↑, ETFA↑, SOD2↑
PRDX6↑, IDH3B↓, PDIA5↑, PRDX3↑, COX5A↓, ETFA ↑, SOD2↑
Caspase inhibitor activity
PRDX6↑, PRDX3↑, SOD2↑
Validation of PRDX3 expression in mitochondria of metastatic and nonmetastatic cells by Western blotting
Efficiency of PRDX3 siRNA transient transfection in mitochondria of metastatic 5-8F cells
Count of metastatic 5-8F cells migrated in Transwell Migration assay
Nasopharyngeal carcinoma (NPC) is a malignant tumor with a high incidence of metastasis, has a great tendency to invade adjacent regions and metastasize to regional lymph node and distant organs. Though the molecular mechanisms of NPC metastasis remain poorly understood, the functions of mitochondria that involved in this process have been uncovered gradually: mitochondria are closely related to oxidative stress process in a cell. Different from the historical views, which assumed oxidative stress contributes to tumor initiation and progression solely by inducing genomic instability, the present views consider oxidative stress directly contributes to tumor metastasis. According to the tumor-stroma co-evolution model, cancer cells induce oxidative stress in adjacent stromal cells and result in an excess production of ROS in mitochondria. Excess stromal ROS induces cytoskeleton rearrangements, autophagy, and mitophagy in the tumor microenvironment to help cancer cells escape excess ROS in the primary tumor site, also induces the stromal over-production of recycled nutrients such as ketones and L-lactate that are fuel for tumor growth and metastasis [10–16]. Moreover, the enhanced cellular antioxidant capability and antioxidant function of redox-signaling in cancer cells result in an increase of cell resistance to oxidant, defend cancer cells themselves from oxidative damage, and favor their metastasis .
In general the balance between ROS generation and elimination is delicately maintained in cells. However, for many cancer cells, an increase in ROS production or decrease in ROS-scavenger capacity disturbs redox homeostasis which leads to an overall increase of intracellular ROS level. Over-expressed catalase to alleviate mitochondrial oxidative stress in a mouse animal model of breast cancer could reduce metastatic tumor burden by >12-fold . Considered this catalase experimental result and our PRDX3 study, it is very likely that a molecular network based on redox-signaling pathways in cancer cells directly contributes to tumor metastasis, this process relates closely to mitochondrial oxidative stress and ROS produced by mitochondria might play an important role in tumorigenesis (its direct action on growth factors and transcription factors) and tumor metastasis as well.
PRDX6 increased about 2.5-fold in mitochondria of metastatic 5-8F cells than in mitochondria of nonmetastatic 6-10B cells. This result was consistent with the results of other research groups: PRDX6 expressions were increased in node-positive breast carcinoma samples compared with node-negative breast carcinoma samples ; enzymatic activities of PRDX6 in lung cancer cells metastasis ; over-expression of PRDX6 led to a more invasive phenotype and metastatic potential in human breast cancer . Over-expression of PRDX6 is on the one hand to strengthen the peroxidase activity of a cell to reduce peroxidants such as H2O2 and hydroperoxides for the relief of oxidative stress. Besides of its peroxidase activity, PRDX6 has additional phospholipase A2 (PLA2) activity, this activity promotes accumulation of arochidonic acid, which, in turn, induces the invasive pathway involving p38 kinase, phosphoinositide 3-kinase, Akt and urokinase-type plasminogen activator. Over-expression of PRDX6 leads to a more invasive phenotype and metastasis potential, at least in part, through regulation of the levels of uPAR, Ets-1, MMP-9, RhoC, and TIMP-2 expression [20, 21]. Therefore, PRDX6 in NPC metastatic process demonstrates a double-side effect. To our knowledge this was the first report of the relationship between PRDX6 and NPC metastasis.
SOD2 increased in many tumor types as they progress from early stage non-invasive disease to late stage metastatic disease, and its over-expression in many instances enhance the metastasis phenotype because SOD2 is able to catalize toxic oxidant O2 .- into another toxic oxidant H2O2 and hydroperoxides, and O2 .- and H2O2 / hydroperoxides both directly contribute to oxidative stress. However, this metastatic phenotype can be reversed by efficient H2O2 scavenging [22–26]. Our proteomic results also showed that SOD2 increased about 3.3-fold in mitochondria of metastatic 5-8F than in nonmetastatic 6-10B cells. To our knowledge this was the first report of the relationship between SOD2 and NPC metastasis.
Over-expression of ECH1 can enhance the energy generation, and promote adhension and migration capability of cancer cell for cancer metastasis. The present experimental data showed that ECH1 increased in 4.1-fold in mitochondria of NPC metastatic 5-8F than nonmetastatic 6-10B cells. This result was consistent with the other studies , a higher expression level of ECH1 was confirmed in tissue from patients with gastric carcinoma with lymph node metastases, and down-regulation of ECH1 could inhibit tumor proliferation, increase the ratio in S phase to G1 phase and decrease the adhesion and migration capacity.
Studies  showed over-expression of each SERPINB gene could effectively suppress the invasiveness and motility of malignant cancer cells. Nuclear localization of SERPINB5 is required for its tumor metastasis suppressor function and it was enriched at the promoter of colony-stimulating factor (CSF-1) in chromatin immunoprecipitation and associated with diminished levels of CSF-1 mRNA. It suggests that its mechanism of action involves, in part, direct association of SERPINB5 with target genes to inhibit metastatic phenotype . The present studies showed a down-regulation of SERPINB5 by 3.2-fold in mitochondria of NPC metastatic 5-8F than nonmetastatic 6-10B cells. Thus, the down-regulated SERPINB5 might enhance the metastatic capability of an NPC cancer cell.
COX subunit composition is regulated by COX5A and COX5B gene transcription in response to high and low O2, respectively. This subunit switch maintains mitochondria redox homeostasis and relates to morphological and functional alteration of mitochondria . PDIA5 also directly involves in the redox homeostasis. The present studies demonstrate that the down-regulation of COX5A and up-regulation of PDIA5 in NPC metastatic 5-8F cells relative to nonmetastatic 6-10B cells. The abnormal expression of COX5A and PDIA5 in NPC metastatic 5-8F cells might disturb the redox homeostasis to contribute to its metastatic process.
Studies  demonstrated over-expression of EIF5A enhances cell motility and promotes tumor metastasis in hepatocellular carcinoma. It was able to induce epithelial-mensenchymal transition (EMT), a key event in tumor invasion and metastasis, characterized by down-regulation of epithelial markers (E-cadherin and beta-catenin) and up-regulation of mesenchymal markers (fibronectin, N-cadherin, alpha-SMA and vimentin). It could also stimulate cytoskeleton rearrangement through activation of RhoA/Rac1 to contribute to the cancer cell metastasis. The present data showed EIF5A increased 2.5-fold in mitochondria of NPC metastatic 5-8F than nonmetastatic 6-10B cells. It suggests that over-expressed EIF5A contributes to NPC cancer cell metastasis.
IDH3B  involves in the mitochondrial tricarboxylic acid cycle that is initiated by acetyl-CoA. Acetyl-CoA is derived from the travel of pyruvic acid from cytoplasm into the interior of the mitochondrion. Pyruvic acid is generated from the glycolysis of glucose. The current study demonstrated the down-regulation of mitochondrial IDH3B in NPC metastatic 5-8F cells. Down-regulated IDH3B will obstacle the tricarboxylic acid cycle to reduce the generation of ATP, cause energy metabolism obstacle and mitochondrial dysfunction, and promote oxidative stress. Meanwhile, the tricarboxylic acid cycle obstacle will built up the pyruvic acid. Studies  demonstrated that pyruvic acid has strong angiogenic activity and plays an important role in angiogenesis for tumor growth and metastasis.
PSMC4 is a 26S protease that is involved in the ATP-dependent degradation of ubiquitinated proteins in the ubiquitin-proteasome system (UPS) that is extensively associated cancer pathogenesis . The UPS plays a central role in fine-tuning the functions of core proangiogenic proteins including VEGF, VEGFR-2, angiogenic signaling proteins such as the PLCy1 and PI3 kinase/AKT pathways, and other non-VEGF angiogenic pathways to regulate the tumor angiogenesis . The up-regulation of PSMC4 would contribute to this angiogenesis process for tumor metastasis.
Moreover, ETFA is a mitochondrial electron transfer flavoprotein . The ETFA up-regulation in NPC metastatic cell could promote mitochondrial electron transport and oxidative phosphorylation, and enhance mitochondrial oxidative stress. Prohibitin (PHB) is a pro-oncogene, inhibits DNA synthesis, and regulates proliferation. However, PHB gene mutated in human cancers , PHB protein responds to mitochondrial stress and may play a role in regulating mitochondrial respiration activity . MRPL12 may act as a scaffolding protein or stabilizer of respiratory chain supercomplexes, is involved in regulation of mitochondrial protein translation and respiration, and plays a role in mitochondrial-mediated death. The current studies found down-regulation of MRPL12, which would regulate mitochondria-mediated cell death and respiration [39, 40]. VDAC1 and VDAC2 forms a channel through the mitochondrial outer membrane with a weak anion selectivity at the open state and a cation-selectivity at the close state [41, 42], and GALU is involved in the binding of calcium ions . The current studies showed the up-regulated expression of VDAC1, VDAC2, and GALU, they would disturb the mitochondrial ion homeostasis in the mitochondrial dysfunction and oxidative stress. The relationship of those mitochondrial DEPs and the tumor metastasis needs to be validated with experiments; however, these data are novel clues to insight into the mechanisms of NPC metastasis.
In summary, the present study provides new insights for exploring the significance of mitochondria and oxidative stress in NPC metastatic processes. Ten mitochondrial DEPs including PRDX3, PRDX6, SOD2, ECH1, SERPINB5, COX5A, PDIA5, EIF5A, IDH3B, and PSMC4 have been rationalized in the tumor-stroma co-evolution model that mitochondrial oxidative stress directly contributes to tumor metastasis (Figure 9). The expression suppression of mitochondrial PRDX3 protein in the anti-oxidative stress system enhanced the mobility potential of NPC metastatic cancer cells, which consolidates the roles of mitochondrial oxidative stress in NPC metastasis. Further investigation is required to determine the biological consequences of all identified mitochondrial DEPs and their relevance to the pathogenic mechanisms of NPC metastasis.
All primary and secondary antibodies, siRNA duplex and a scrambled sequence control siRNA were from Santa Cruz. LipofectamineTM RNAiMAX transfection reagents and serum-free medium were from Invitrogen. Immobiline DryStrip gels (pH 3-10 NL, 24 cm), CyDye DIGE fluor Cy2, Cy3 and Cy5 were from GE Healthcare. Matrigel basement membrane matrix was from BD Biosciences. Transwell plates and inserts (Cat. No. 3422) were from Corning. All other chemicals, unless stated otherwise, were obtained from Sigma.
NPC 5-8F and 6-10B cells were obtained from the Modern Analytical Testing Center of Central South University of China. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics in a humidified incubator with 5% CO2 in air at 37°C. The cells were grown to 80% confluence, washed twice with D-Hank’s solution, digested with 0.25% trypsin and 0.02% ethylenediaminetetraacetate dehydrate (EDTA), collected by centrifugation (400 × g, 10 min, 4°C). A total of ~2 × 107 cells were needed for experiments.
Preparation and characterization of mitochondria, and extraction of mitochondrial proteins
Mitochondria were isolated from 5-8F and 6-10B cells, respectively, according to the Sucrose Gradient Separation Manual (http://www.mitosciences.com) with a minor modification. Briefly, the collected cells described above were resuspended in five volumes of Mito buffers (0.25 M sucrose, 1 mM EDTA, 10 mM Tris·HCl pH 7.4, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mg/ml leupeptin, 1 mg/ml pepstatin) and homogenized by pre-cooled Potter-Elvehjem homogenizer (1,000 rpm; around 20 up and down strokes). The homogenates were centrifuged (1,000 × g, 10 min, 4°C) to collect the supernatants. The supernatants were centrifuged (12,000 × g, 15 min, 4°C) to collect the pellets. The pellets were resuspended in 0.5 ml Mito buffers. The discontinuous sucrose gradient separation solutions were prepared in centrifuge tubes with gradient from bottom to top that is 0.5 ml of 2.0 M sucrose, 1 ml of 1.3 M sucrose, 1 ml of 1.0 M sucrose, and 0.5 ml of 0.8 M sucrose. The resuspended samples were carefully applied to the top of gradient solutions and centrifuged (80,000 × g, 2 h, 4°C; Beckman swinging bucket 55 Ti rotors). The brown belts between 1.0 M and 1.3 M sucrose layers were collected carefully with a 5-ml syringe needle immediately after centrifugation. A volume (600 μl) of methanol was added to the top of 150 μl brown sucrose solution, and mixed; then 150 μl of chloroform and 450 μl of water were added, mixed again, and centrifuged (18,000 × g, 5 min, 4°C). All organic and inorganic liquids in centrifuge tubes were poured off carefully, followed by air dry at 4°C of the white discs of proteins. The white discs of proteins were solubilized (2 h; intermittent mix) in 800 μl lysis buffer that contained 8 M urea, 2 M thiourea, 130 mM dithiothreitol (DTT) , 4% 3-[(3-cholanidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), 40 mM tris base, 1 mM EDTA, 1 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml pepstatin, and pH 8.5 adjusted by 50 mM NaOH, and centrifuged (18,000 × g, 2 h, 4°C). The supernatants were collected. The protein concentration of the supernatants was determined by a 2-D Quantification kit (GE Healthcare). The isolated mitochondria were characterized according to Murayama’s method . Briefly, the extracted mitochondrial proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to polyvinylidene fluoride (PVDF) membranes (Semi-dry Nova blot, Pharmacia Biotech), probed with various primary antibodies against marker proteins from different cellular compartments (LDH, PCNA, COX IV, catalase, GRP 78, and cathepsin D) and corresponding secondary antibodies, and visualized by a SuperSingal West Pico ECL kit (Thermo). Preparation of isolated mitochondria for electron microscopic observation was carried out according to the related manual and observation was made by a Hitachi H 7500 transmission electron microscope.
2D-DIGE and imaging analyses
MS-identification of mitochondrial DEPs
The 2D gel spots that contained DEPs were excised from the preparative gels. The proteins in the gel-spot were destained, reducted, alkylated, and digested in gels with trypsin . For PMF analysis, the tryptic peptides were eluted from ZipTips with 80% acetonitrile/0.1% trifluoroacetic acid, and were spotted onto a MALDI target with an equal volume of cyano-4-hydroxycinnamic acid (10 mg/ml; Sigma) saturated with 50%acetonitrile in 0.05% TFA. The mass spectrometric (MS) spectra were acquired on a Voyager DE STR MALDI-TOF mass spectrometer (ABI, Foster City, CA). The instrument settings were reflector mode with 160-ns delay extraction time, positive polarity, and 20-kV accelerating voltage. The spectrum mass-tocharge (m/z) were usually acquired with laser shots at 200/spectrum and ranged from 1000 to 4000 m/z. External calibration was performed using a Peptide Mass Standard kit (Perspective Biosystems, Framingham, MA). The acquired MS spectra were processed with base-line correction, noise removal (5%), and peak deisotoping using DataExplore (Version 188.8.131.52) software to generate the peaklist for peptide mass fingerprint (PMF) analysis. The PMF data were input into Mascot (http://www.matrixscience.com/) for protein identity searching. The PMF data from the Voyager DE STR mass spectrometer were searched against the NCBInr database (release date January 7, 2012; 16,831,682 sequences, 5,781,564,572 residues; Homo sapiens 241,934 sequences). The search was restricted to “Human” as taxonomy. The other search parameters included type of search (peptide mass fingerprint), enzyme (trypsin), fixed modifications (carbamidomethylcysteine), variable modifications (methionine oxidation), mass values (monoisotopic), protein mass (unrestricted), peptide mass tolerance (100 ppm), peptide charge state (1+), and 1 maximum missed cleavages. Proteins whose scores were greater than 66 were considered as significance (p < 0.05), and only human proteins with the best score in each Mascot search were accepted as successful identifications. The coverage of amino acid sequence, the Mascot score, the mass and pI, and accession number are obtained for each protein.
Proteins in a 2D gel spot that was not identified or was a mixture identified by MALDI-TOF PMF data were subjected to electrospray ionization (ESI)-quadrople (Q)-TOF MS/MS analysis. Briefly, the tryptic peptides from 2D gel spots were loaded onto a C18 pre-column for concentrations and fast desalting, and then eluted to the reversed-phase column for separation. MS/MS spectra were performed in data-depended mode in which up to four precursor ions above an intensity threshold of 7 counts/seconds (cps) were selected for MS/MS analysis from each survey scan. For MS/MS database query, the peptide sequence tag (PKL) format file that was generated from MS/MS data with MassLynx v 4.0 software were inputted into the Mascot search engine to search protein against the NCBInr database (release date January 7, 2012; 16,831,682 sequences, 5,781,564,572 residues; Homo sapiens 241,934 sequences). A mass tolerance of 0.3 Da for both parent (MS) and fragmented (MS/MS) ions, allowance for up to one trypsin miscleavage, variable amino acid modifications consisting of methionine oxidation and cysteine carbamidomethylation were used. MS/MS ion score threshold was determined to produce a false-positive rate less than 5% for a significant hit (P < 0.05). The false-positive rate was calculated with 2∗ reverse/(reverse + forward)/100. In the current study, the MS/MS ion score threshold was 45 and a false-positive rate was approximately 3.1%. Only protein IDH3B was identified with only one peptide with a Mascot score of 73, its MS/MS spectrum was further checked manually and interpreted with de-novo sequencing.
Functional enrichment analyses of mitochondrial DEPs and protein-protein interaction data
The gene names of mitochondrial DEPs were converted to NCBI-Entrez format for consistency and saved as a text file that was inputted into Cytoscape v 2.8.2 (http://www.cytoscape.org), and BiNGO plugin 2.44 downloaded from Cytoscape manage plugin was used to analyze the enriched biological processes and molecular functions. Human protein-protein interaction data that were downloaded from HPRD (release 9; http://www.hprd.org) and NCBI databases (http://www.ncbi.nlm.nih.gov) were used to generate a protein-protein interaction sub-network of mitochondrial DEPs; the result only extended one layer connection and could be visualized by Cytoscape.
siRNA transient transfection
The siRNA transient transfections of metastatic 5-8F cells were conducted according to siRNA Transfection Protocols (Santa Cruz) and Transfection Technology Protocols (Invitrogen) with a minor modification. Briefly, the cells were incubated in a six wells plate (Costar) at 37°C in a CO2 incubator till 70-80% confluence. The solution A (2.4 μl RNAiMAX in 100 μl serum-free medium) and solution B (10 μl siRNAs duplex in 100 μl serum-free medium) were mixed gently (the final concentration in each well was 100 nM), and incubated (room temperature; 45 min). Cells were washed twice with serum-free medium, and then added into 200 μl of above A + B mixture and 800 μl of serum-free medium in each well. After cells were incubated (7 h), the medium in each well were removed and replaced with 2.5 ml normal fresh growth medium, cells were continued to be incubated (48 h). Cells were washed twice with D-Hank’s solution, digested with 0.25% trypsin and 0.02% EDTA, collected by centrifugation (400 × g; 10 min; 4°C). A scrambled sequence control siRNA experiment was conducted at the same time. The efficiency of siRNA transfection was tested by Western blotting.
Transwell Migration assays
Cells Transwell Migration assay was carried out according to Matrigel Basement Membrane Matrix Product Specification Sheet (BD Biosciences) and Transwell Insert Product Description (Corning) with a minor modification. Briefly, the Matrigel basement membrane matrix was thawed (4°C; overnight), the Transwell plates was put and inserted (both were added with serum-free medium) in a CO2 incubator (37°C; 2 h). The medium in Transwell plates was removed and Transwell plates were inserted and kept on ice. Pre-cooled pipettes were used to add 20 μl Matrigel matrix (1:1 diluted with pre-cooled serum-free medium) into each compartment of Transwell inserts and swirled immediately to disperse materials evenly, placed Transwell inserts (37°C; 30 min) to form thin Matrigel. A volume (600 μl) of medium (RPMI 1640 with 10% FBS and 5 μg/ml fibronectin) was added to each Transwell plates well, followed by adding the above thin Matrigel inserts, then 200 μl medium and the cells (resuspended cells in medium at a density of 5 × 104 cells/ml) were added into each compartment of thin Matrigel inserts, and incubated in a CO2 incubator (37°C; 24 h). All of these experiments should be carefully handled using sterile techniques. Those non-migrated cells on each compartment of thin Matrigel inserts were scraped off with cotton swabs, the migrated cells were fixed and stained by 0.1% crystal violet solution. The cells without siRNA transient transfection and with a scrambled sequence transfection were used as control groups. In three independent experiments, the number of cells that migrated through Transwell insert membrane was counted in 9 visual fields per well at 400 × under an Olympus microscope, means of these counts were calculated for statistical analysis using SPSS software, and variables were compared using Student’s t-test with a significance level of P = 0.05.
Differentially expressed gene
Differentially expressed protein
Matrix-assisted laser desorption ionization
Tandem mass spectrometry
Peptide mass fingerprint
Reactive oxygen species
Two-dimensional difference in-gel electrophoresis
This work was supported by the National Basic Research Program of China (Grant No. 2011CB910704), the National Natural Science Foundation of China (Grant No. 30973289), and Xiangya Hospital Funds for Talent Introduction (X.Z.).
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