Screening Of Downregulated Genes Related To The Occurrence Of Nasopharyngeal Carcinoma And The Expression Of Annexin A6in Nasopharyngeal Carcinoma

Objective:To discover the downregulated genes related to the occurrence of nasopharyngeal carcinoma, the technology of quantitative proteomics was used to identify downregulated differentially expressed proteins among immortalized nasopharyngeal epithelial cell line (NP69) and nasopharyngeal carcinoma cell lines (6-10B and5-8F).To study the expression of ANXA6in the nasopharyngeal carcinoma and the methylation of the gene in5-8F,6-10B and NP69,in order to primarily discuss the mechanism of down-regulation expression and role of ANXA6in nasopharyngeal carcinoma.Methods:In this study, iTRAQ-tagging combined with2DLC-MS/MS analysis and ProteinPilotTM4.2software were used to identify downregulated proteins in Swissprot protein database among5-8F,6-10B and NP60cell lines. Bioinformatic analysis was performed in these differentially expressed proteins. Western Blotting and Quantitative PCR were firstly used to detect the expression of ANXA6in5-8F,6-10B and NP69on protein and mRNA levels respectively. Immunohistochemistry was used to detect the expression of ANXA6between NPC tissues and normal nasopharyngeal epithelial tissues and analysis of ANXA6protein expression in clinicopathological features. In addition, Bisulfite Sequencing PCR was performed to analyze methylation of ANXA6CpG sites in these three cell lines. SPSS17.0statistical software was used for the data analysis.Results:According to the selected criteria,98down-regulated differentially expressed proteins were identified. Through the bioinformatic analysis, these proteins were found to play a role in the occurrence of NPC through a variety of signaling pathways.The mRNA and protein levels of ANXA6were gradual decline in NP69,6-1OB and5-8F cell lines which was the same as quantitative proteomics. Immunohistochemistry showed that expression of ANXA6was decreased in NPC tissues compared with normal nasopharyngeal epithelial tissues, and was associated with lymph node and distant metastasis and clinical stage. Additionally, BSP showed that the promoter region of ANXA6gene was nearly non-methylated in NP69cell line, and was partly methylated in5-8F and6-1OB cell lines with no statistically significant methylated percentages25.6%and17.9%.Conclusions:1.98down-regulated differentially expressed proteins are identified by using iTARQ combined with2DLC-MS/MS in NP69,5-8F and6-10B cell lines.2. The expressions of protein ANXA6are gradually decreased in NP69,6-10B and5-8F cell lines,which are the same as in nasopharyngeal epithelial tissue,non metastasis and metastasis NPC tissues. It could be inferred that the down-regulation of ANXA6may be related with the occurrence and development of NPC.3. The loss/down-regulation of ANXA6expression is closely correlated with distant metastasis, lymph node metastasis and clinical stage. NPC with loss/down-regulation of ANXA6expression are more likely to occur with distant metastasis, lymph node metastasis and often has later clinical stage.4. The loss/down-regulated expression of ANXA6in NPC may partly due to its methylation. Moreover, other mechanisms may participate in the expression of ANXA6at the level of transcription.5.ANXA6may be a tumor suppressor gene in NPC.8Figures,8tables,53references...