The Study On The Relationship Between Methylation Of MicroRNAs And Esophageal Squamous Cell Carcinoma

ObjectivesTo search the microRNAs that is regulated potentially by DNA methylation inesophageal squamous cell carcinoma and screen the differential expression ofmicroRNAs in esophageal squamous cell carcinoma, to look for new biologicalmarkers for esophageal squamous cell carcinoma from the standpoint of epigenetics,which in order to investigate the etiopathogenesis of esophageal squamous cellcarcinoma from molecular level, provide new thought and theory evidence for theearly diagnosis and prognosis of esophageal squamous cell carcinoma and supply newtherapeutic targets for esophageal squamous cell carcinoma.Methods1. The CpG islands of microRNAs were predicted by the bioinformatics method,including miRBase database, UCSC Genome Browser, CpG Island Searcher andEuropean Bioinformatics Institute website and so on.2. The relative quantifications of microRNAs between esophageal squamous cellcarcinoma cell lines and normal esophageal squamous epithelial cell line, betweenesophageal squamous cell carcinoma cell lines and normal esophageal squamousepithelial tissue, and between esophageal squamous cell carcinoma cell lines with5-Aza-2’-deoxycytidine and without were detected by the Real-time quantitativeReverse-Transcription Polymerase Chain Reaction (Poly A method) and were used thetechnology of miProfileTMmiRNAs qPCR microarray in high throughout. Screeningthe miRNAs of the fold changes (tumor cell lines vs. normal esophageal squamousepithelial cell line) and the fold changes (tumor cell lines vs. normal esophagealsquamous epithelial tissue)＜0.5and the fold changes (tumor cell lines with5-Aza-2’-deoxycytidine vs. tumor cell lines without5-Aza-2’-deoxycytidine)＞2. 3. The△Cts of4esophageal squamous cell carcinoma cell lines, normal esophagealsquamous epithelial cell line and normal esophageal squamous epithelial tissue werelogarithmic transformed and then were drawed a heatmap and cluster analysed by Rsoftware.4. The relative quantifications of15microRNAs between tumor tissues and adjacentnormal tissues in10patients of esophageal squamous cell carcinoma were detected bythe Real-time quantitative Reverse-Transcription Polymerase Chain Reaction (Poly Amethod). To every microRNAs, the paired t-test was used to compare the△Ctbetween tumor tissues and adjacent normal tissues.Results1. After the prognosis of bioinformatics, there were1527microRNAs in human and88microRNAs that is regulated potentially by DNA methylation.2. After the cellular experiments, We found that there were15microRNAs that isregulated potentially by DNA methylation, including hsa-miR-9-5p, hsa-miR-203,hsa-miR-375, hsa-miR-1258, hsa-miR-1914-5p, hsa-miR-3665, hsa-miR-4470,hsa-miR-4479, hsa-miR-4530, hsa-miR-4634, hsa-miR-4664-5p, hsa-miR-4665-5p,hsa-miR-4674, hsa-miR-4687-5and hsa-miR-4734.3. The result of cluster analysis: normal esophageal squamous epithelial cell lineHet-1A and normal esophageal squamous epithelial tissue were in the same category,4esophageal squamous cell carcinoma cell lines were in the same category, which candistinguish esophageal squamous cell carcinoma and normal cell and tissue.4. Comparing the relative quantifications of15microRNAs between tumor tissuesand adjacent normal tissues in10patients of esophageal squamous cell carcinoma, thenumber of the fold changes＜0.5of hsa-miR-3665and hsa-miR-375were7and6,respectively. And the relative quantification of hsa-miR-3665was significant(t=2.564，P=0.030).Conclusions1. The relative quantifications of microRNAs that are regulated potentially by DNAmethylation can distinguish esophageal squamous cell carcinoma and normal cell and tissue.2. In our study, hsa-miR-3665and hsa-miR-375may be two miRNAs which areregulated potentially by DNA methylation and are down-regulated in esophagealsquamous cell carcinoma.