| Objective:Lung cancer is one of the highest morbidity of malignant tumor in China, and Xuanwei, Yunnan province is one of the area where is the highest lung cancer incidence in China. Lung cancer in Xuanwei is with a poor prognosis, from 2011 to 2013, the mortality rate was 116.35/100,000 and 92.00/100,000 for male and female respectively, the lung cancer is account for 63.03% of the total cancer mortality. The pathogenesis of it has remain been unclear, it is significant to study the pathogenesis of lung cancer in xuanwei for early diagnosis and effective drug treatment to improve the prognosis of patients with lung cancer. DNA methylation is an important part of epigenetics, most studies have shown that the differential methylation occurrence in tumorigenicity and hypermethylation can be reversed by methyl transferase inhibitors which provided new opportunities to early diagnosis and targeted therapies for cancer. High throughput technologies such as methylated DNA immunoprecipitation combine with microarray analysis (MeDIP-chip) and precise techniques such as mass spectrometry technology developed to provide a new platform for understanding the genome-wide distribution of abnormal methylation and the methylation level of specific sites.Method:1. Differentially methylated regions (DMRs) and differentially expressed genes (DEGs) were detected by MeDIP-chip and expression profiling respectively in lung cancer cases (n=10). Integrated analysis the result of MeDIP, expression profiling, the gene ontology (GO) and pathway analysis to find out the candidate genes which were more hypermethylation, lower expression and tumor specific.2. More lung cancer cases in Xuanwei (n=45) tested by methylation-sensitive high resolution melting curve (MS-HRM) to validate the methylated status of the sequences. And we detected the methylated level of each CpG unit of candidate seqences precisely by mass spectrometry technology in lung cancer cases in xuanwei (n=36).To verify the expression level of candidate gene we detected by real-time fluorescent quantitative PCR (RT-qPCR) and western blot in lung cancer cases (n=39, n=35, respectively).3. The results of RT-qPCR and mass spectrometry were grouped by clinical data of lung cancer patients in Xuanwei and then comparative analysis, survival curve analysis were performed in groups via using SPSS22.0 statistical software.Result:1. It found 21,432 DMRs through MeDIP-chip between lung cancer and matched non-cancerous lung tissues, it had 5,788 hypermethylated regions and 1111 hypomethylated regions after defined log ratio, and 4,452 regions of them were in the promoter, accounted for 64.5%(4452/6899)of the abnormal methylation, these DMRs mainly distributed in chromosome 1,11,16,17, and 19. Finally,58 regions which were in the promoter, CGIs, more hypermethylation and downregulated in more samples and which belonged to 38 genes were screened out, most of these genes in lung cancer has never been reported on our search scope. In this study, we chose 4 DMRs eventually which had more hypermethylation, downregulation in more samples and closed to the transcription start site as candidate sequences for further validation, the candidante genes were STXBP6, BCL6B, FZD10 and HSPB6.2. In order to validate the methylation and expression level of the 4 candidate genes, first of all, we used MS-HRM and mass spectrometry to detect the methylation status in more lung cancer cases in Xuanwei, the result of detected by MS-HRM in 45 lung cancer cases showed that the proportion of lung cancer tissues with hypermethylation was significantly higher than that of adjacent tissue (STXBP6:55.6% vs. 20%, BCL6B:68.9% vs.40%, FZD10:93.3% vs.77.8%, HSPB6:91.1% vs.68.9%), the differences were statistically significant (P<0.05), So did the result of tested by mass spectrometry, there were statistically significance (all P<0.05). Second, the mRNA level of STXBP6, BCL6B, FZD10 and HSPB6 were downregulation in most lung cancer which accouted for 66% or more of all cases detected by RT-qPCR analysis, the difference of mRNA level of candidate genes between the two groups were significantly statistic (P<0.001). Finally, so did the result of detecting by western blot, the downrelation were more in the expression level of protein than that of mRNA, accounted for 88.57% or more of lung cancer tissues for each candidate genes. The differences of the protein level of candidate genes between the two groups were statistically significant (P<0.001).3. The result of mass spectrometry grouped by the clinical data of patients and compared within groups. The methylation level of CpG20 and CpG21 units of STXBP6 were higer in clinical stage III than in I and II in lung cancer tissues (P=0.013, P=0.033). CpGl and CpG38 units of FZD10 were more hypermethylation in clinical stage I than in advanced clinical stage in lung cancer tissues (P=0.049, P=0.042). CpG9 unit of BCL6B had more hypermethylatin in the lung cancer patients were with lymph node metastasis (P=0.035). As far as mRNA, there were no correlation between the expressive status of candidate genes and clinicopathological features such as gender, smoking, pathological stage, lymph node metastasis, prognosis, while mRNA level of them were found to be associated with age of patients. The 4 genes downregulation obviously in tumor tissues were in younger patients who were less than 45 years old (P<0.05).Conclusion:DMRs and DEGs in this study provides important informations to understand the pathogenesis of lung cancer in Xuanwei and find diagnostic and therapic biomarkers for it. STXBP6, BCL6B, FZD10 and HSPB6 may be potential methylation markers used for diagnosis early, treatment and prognosis of lung cancer in Xuanwei. |
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