MethylCap-seq Based DNA Methylation Profiling Of Novel Candidate Genes And Clinical Value In Clear Cell Renal Cell Carcinoma

Part I:MethylCap-seq data and screening of DNA methylation in cell line786-0Objective:To screen and validate different methylated regions in cell line786-0. Methods:MethyCap-seq data was supplied by Dr. Guo Shicheng (Fudan University).786-0was cultured in RPMI1640. One case of adjacent normal tissue was extract from the Tissue Bank of Shanghai Cancer Center. DNA was isolated by kit and the status of methylation was test by BSP. Result:The methylation rate of CD9was84.29%in adjacent normal tissue VS88.57%in786-0. The methylation rate of FBXW10was22.22%in adjacent normal tissue VS84.44%in786-0. The methylation rate of HIST1H3E was97.33%in adjacent normal tissue VS96%in786-0The methylation rate of LEP was67.03%in adjacent normal tissue VS84.32%in786-0The methylation rate of SMPD3was35.71%in adjacent normal tissue VS82.86%in786-0The methylation rate of GGT6was94.29%in adjacent normal tissue VS80%in786-0. Conclusion:The results showed that hypermethylation of FBXW10and SMPD3occurred only in cell line786-0. Part Ⅱ:Validation of methylation profiling of SMPD3and FBXW10in tissuesObjective:To validate the difference of methylation rate of FBXW10and SMPD3in tumor tissue and adjacent normal tissue. Methods:Paired tissue samples were retracted from the Tissue Bank of Shanghai Cancer Center. DNA was isolated as before. Bisulfite converted DNA and then tested the methylation statu by Pyroseqencing. Result:85cases were obtained. Methylation rate of SMPD3was48.78%in tumor tissue VS34.62%in adjacent normal tissue (p<0.001). And methylation rate of FBXW10was58.98%in tumor tissue VS38.66%in adjacent normal tissue (p<0.001). Conclusion: Methylation rate of FBXW10and SMPD3in tumor tissue was higher than that in adjacent normal tissue. Part Ⅲ:clinical value of methylation profiling of SMPD3and FBXW10Objective:To study the clinical value of methyaltion rate of SMPD3and FBXW10. Methods:Collected clinical data of Fuhrman grade, tumor size, tumor stages and prognosis. The correlations of DMRs with Fuhrman grade, tumor size, tumor stages and prognosis were confirmed. Results:Only in T1stage RCC cohort, methylation rate of RCC tissue was correlated with furhman grade, furhman grade Ⅰ、Ⅱ、Ⅲ is separate51.89%,60.37%and66.18%(p=0.020). There is significant higher methylation rate in normal tissue when furhman grade is lower (p=0.004). ROC confirmed that26.4%was the optimal threshold value to predict survival. Kaplain-Meir analysis showed that hypermethylation group survived longer than hypomethyaltion group with a significant p vavle0.04. Conclusion: FBXW10was a useful biomarker to predict Fuhrman grade in T1stage tumor and improved the accuracy of percutaneous renal biopsy. SMPD3hypermethylation of adjacent normal tissue was a useful specific biomarker to predict poor prognosis. Part Ⅳ: expression and methylated inactivation of SMPD3and FBXW10Objective:To study the expression of SMPD3and FBXW10before and after demethylation treatment。Methods:RNA was isolated by TRIZOL. Gene expression before and after5’-Aza treatment was studied by Real-time PCR. Results:The qRT-PCR analysis demonstrated that both SMPD3and FBXW10mRNA expression of was upregulated after5-Aza-20-deoxycytidine treatment in cell line. SMPD3rose2.47times and FBXW10rose1.67times. Conclusion:Two DMRs which located in SMPD3and FBXW10were found to be hypermethylation in ccRCC and participant hypermethylated inactivation...