Screening And Functional Study Of Methylation Sites Of Hepatocellular Carcinoma(HCC) Related NcRNA Promoter

Part 1:Screening and functional study of methylation sites of HCC related miRNA promoter.Objective:Previous studies have found the promoter methylation of the coding gene plays important roles in the occurrence and development of HCC.Promoter methylation of tumor suppressor gene is the direct cause of its low expression in HCC.Similarly,the abnormal promoter region methylation pattern of encoding miRNA gene is also closely related to the occurrence and development of HCC.Therefore,this study investigates the effect of miRNA promoter methylation in HCC and its effects on the mechanism of the occurrence and development of HCC.The miRNA promoter methylation sites that is associated with overall survival,vascular invasion,pathological grade,and clinical stage of HCC were screened and verified by bioinformatics.Which has provided basis for further research.Methods:1.All the data including Clinical data(Clinical),miRNA expression profiling data(microRNA),DNA methylation data(Methylation),and mRNA expression profiling data(mRNA)were obtained from TCGA database.The "minfi" package in the R language was used to analyze the difference in the Beta values of these miRNA promoter methylation sites in HCC cancer tissues and adjacent tissues.Screening for methylation sites with a q value<0.05 and an absolute value>0.1 for the Beta difference between HCC and adjacent tissues;The methylated sites above were then analyzed for their correlation with miRNA expression,and the methylated sites were screened out for their significant negative correlation with miRNA expression.The methylation site located within 2KB of miRNA promoter region(2000bp upstream of TSS)was further screened from the methylation site above;2.LASSO-COX algorithm was used to construct the miRNA promoter methylation model of overall survival of patients with HCC;LASSO-logistic was used to construct the miRNA promoter methylation model of vascular invasion,pathological grading and clinical stage of patients with HCC.The foregoing models were used to judge the overall survival,vascular invasion,pathological grading and clinical stage of HCC patients respectively.The accuracy of the models was evaluated by ROC curve analysis and survival analysis;3.Functional analysis was conducted with the miRNAs corresponding to the methylation sites of the miRNA promoter specific for vascular invasion;4.The cancer tissues of 146 patients with HCC who had undergone surgical resection at the First Affiliated Hospital of Wenzhou Medical University from June 1,2016 to April 2018 were divided into vascular infiltration group and non-vascular infiltration group according to their pathological results.Taking the specific vascular infiltrating miRNA promoter methylation sites as an example,the methylation-specific PCR method was used to verify whether the methylation levels of the aforementioned methylation sites were consistent with the results of our previous screening.And verify the accuracy of the aforementioned model;5.The vascular infiltration specific mir-199a-3p mimic was transfected into two HCC cell lines,Huh7 and HCCLM3.The proliferation of the HCC cell lines before and after the transfection was observed by RTCA technique.The Transwell chamber migration assay was used to observe the invasive and transfer ability of the HCC cell lines before and after the transaction.Results:1.A total of 45 miRNA promoter methylation sites with different methylation levels in HCC and paracancerous tissues were screened out;2.The miRNA promoter methylation model constructed to predict the overall survival of HCC patients contains 8 miRNA promoter methylation sites.According to the calibration curve analysis,the c-index of the model was 0.605 at 1 year,0.608 at 3 years,and 0.611 at 5 years;tdROC curve analysis found that the AUC of the model in patients with HCC at 1 year,3 years,and 5 years was 0.624,0.647 and 0.638,respectively;survival curve analysis found that the model can divide the HCC patients into high-risk and low-risk groups(P<0.0001).And after stratification according to the clinical features,the model can also divide most patients in high-risk and low-risk groups(P<0.05);Univariate and multivariate COX regression analysis found that the model and tumor T stage were the separate risk factors for OS in HCC patients,and the combination of the two risk factors has higher accuracy;3.The constructed miRNA promoter methylation model for predicting HCC vascular invasion,pathological grading and clinical stage contained 7,10 and 9 miRNA promoter methylation sites,respectively.The ROC curve analysis found that the AUC of the above three models were 0.667,0.745 and 0.725;4?The target genes of miRNAs corresponding to the 7 miRNA promoter methylation sites related to vascular invasion were involved in various biological processes associated with malignancies(including HCC),such as WNT,MAPK,mTOR signaling pathway,etc;5?After tissue experiment,we found that the differences in methylation levels of the 7 miRNA promoter methylation sites associated with vascular invasion between the vascular infiltration group and the non-vascular infiltration group were consistent with our previous bioinformatics analysis.After transfection of mir-199a-3p mimic into Huh7 and HCCLM3 cells,the proliferation capacity of Huh7 cells was weakened,and the invasion and migration capacity of both Huh7 cells and HCCLM3 cells were weakened.Conclusion:The miRNA promoter methylation sites we screened out can be used to predict the overall survival rate of HCC patients.In addition,it can be used as a biomarker for vascular invasion,tumor pathological grade and clinical stage of HCC.Also,it provides new targets for improving the treatment and prognosis of HCC.Part 2:Screening and functional study of methylation sites of HCC related IncRNA promoter.Objective:LncRNA is another type of ncRNA besides miRNA,which is closely related to the development and progression of tumors.It 15 a rising star in the field of tumor biology in recent years.During the occurrence and development of HCC,abnormal methylation patterns in promoter regions of IncRNA genes were also very important.Therefore,we studied the role of IncRNA promoter methylation in HCC and the molecular mechanisms involved in the development of HCC.The IncRNA promoter methylation sites that were associated with overall survival,vascular invasion,pathological grade,and clinical stage of HCC were screened and verified by bioinformatics.Which has provided basis for further research.Methods:1.All the data including Clinical,Transcriptome File,Methylation and mRNA were all downloaded from the TCGA database and sequenced According to the methylation site annotation of HumallM4ethylation450 chip by TCGA,methylation sites related to lncRNA were screened out(within 2KB of lncRNA promoter region);The beta values of these IncRNA promoter methylation sites in HCC and adjacent tissues were also analyzed by the"minfi" package in the R language;The methylation loci above were analyzed for their correlation with lncRNA expression,and the methylation loci with significant negative correlation between beta and IncRNA expression were screened out;2.LASSO-COX algorithm was used to construct the lncRNA promoter methylation model of overall survival of patients with HCC;LASSO-logistic was used to construct the IncRNA promoter methylation model of vascular invasion,pathological grading and clinical stage of patients with HCC.The foregoing models were used to judge the overall survival,vascular invasion,pathological grading and clinical stage of HCC patients respectively.The accuracy of the models was evaluated by ROC curve analysis and survival analysis;3.Functional analysis was conducted with the lncRNAs corresponding to the methylation sites of the IncRNA promoter specific for vascular invasion;4.The cancer tissues of 146 patients with HCC who had undergone surgical resection in the First Affiliated Hospital of Wenzhou Medical University from June 1,2016 to April 2018 were divided into vascular infiltration group and non-vascular infiltration group according to the pathological results.Taking the specific vascular infiltrating miRNA promoter methylation sites as an example,the methylation-specific PCR method was used to verify whether the methylation levels of the aforementioned methylation sites were consistent with the results of our previous screening.And verify the accuracy of the aforementioned model.5.RNA interference was used to silence the vascular infiltration specific PVT1 in Huh7 and HCCLM3.The proliferation of the HCC cell lines before and after the silence was observed by RTCA technique.The Transwell chamber migration assay was used to observe the invasive ability of the HCC cell lines before and after the silence.Results:1.A total of 113 IncRNA promoter methylation sites with different methylation levels in HCC and paracancerous tissues were screened out;2.The IncRNA promoter methylation model constructed to predict the overall survival of HCC patients contains 10 IncRNA promoter methylation sites.According to the calibration curve analysis,the c-index of the model was 0.675 at 1 year,0.653 at 3 years,and 0.651 at 5 years;tdROC curve analysis found that the AUC of the model in patients with HCC at 1 year,3 years,and 5 years was 0.709,0.637 and 0.679,respectively;survival curve analysis found that the model can divide the HCC patients into high-risk and low-risk groups(P<0.0001).And after stratification according to the clinical features,the model can also divide most patients in high-risk and low-risk groups(P<0.05);Univariate and multivariate COX regression analysis found that the model and tumor T stage were the separate risk factors for OS in HCC patients,and the combination of the two risk factors has higher accuracy;3.The constructed IncRNA promoter methylation model for predicting HCC vascular invasion,pathological grading and clinical stage contained 8,22 and 13 lncRNA promoter methylation sites,respectively.The ROC curve analysis found that the AUC of the above three models were 0.657,0.797 and 0.724;4.The target genes of IncRNAs corresponding to the 8 IncRNA promoter methylation sites related to vascular invasion were involved in various biological processes associated with malignancies(including HCC),such as PI3K-AKT signaling pathway,etc;5.After tissue experiment,we found that the differences in methylation levels of the 8 IncRNA promoter methylation sites associated with vascular invasion between the vascular infiltration group and the non?vascular infiltration group were consistent with our previous bioinformatics analysis.When si.pvtl was transfected into Huh7 and HCCLM3 cells,the proliferation,invasion and migration capacity of both the two cell lines were weakened.Conclusion:The IncRNA promoter methylation sites we screened out can be used to predict the overall survival rate of HCC patients.In addition,it can be used as a biomarker for vascular invasion,tumor pathological grade and clinical stage of HCC.Also,it provides new targets for improving the treatment and prognosis of HCC.