Construction Of DNMT3B Gene RNAi Lentiviral Vector And Its Effect On Migration,Invasion And EMT Of Human Bladder Cancer Cells

BackgroundAs a common malignant tumor of urinary system,bladder cancer is characterized by complex,multi-step and multi-factor pathological changes,which are closely related to gene structure changes and epigenetic studies.DNA methylation,as one of the epigenetic gene modification methods,is involved in the occurrence,development and drug resistance of tumors.DNMT3B,as a significant de novo methylation transferase,is thought to be involved in the regulation of bladder tumor initiation and progression.RNA interference(RNAi)has been widely used in the study of gene function and disease diagnosis and treatment through gene silencing,but there are few reports on the effect of RNAi on the expression of DNMT3B gene on the biological behavior of human bladder cancer cells.In addition,epithelial-mesenchymal transition(EMT)refers to the basic morphogenetic process in which epithelial cells acquire mesenchymal phenotypic characteristics,and is one of the significant mechanisms of tumor cell invasion and metastasis.At present,whether DMNT3B is involved in the regulation of invasion,migration and EMT of human bladder cancer cells needs systematic and in-depth study.ObjectiveDNMT3B gene RNAi lentivirus vector was constructed and transfected into human bladder cancer cells,and the invasion,migration and EMT characteristic protein expression levels of bladder cancer cells after DNMT3B gene silencing were detected,to explore the role and mechanism of DNMT3B in regulating migration,invasion and EMT of bladder cancer.It provides experimental and theoretical basis for DNMT3B as molecular target for human bladder cancer therapy,and has important clinical potential value.Method1.Human bladder cancer EJ cells and UMUC3 cells were selected and transfected with lentivirus vector containing shRNA of DNMT3B gene to construct DNMT3B silent bladder cancer cells,which was called shRNA group.EJ cells and UMUC3 cells were transfected with empty lentivirus as negative control group(NC group).DNMT3B mRNA expression level in shRNA group and NC group was detected by RT-PCR,DNMT3B silencing efficiency was evaluated,and stable transfected cell lines were identified.2.Cell scratch assay and Transwell cell assay were used to detect migration and invasion of shRNA and NC cells.Western Blot was used to detect the expression changes of EMT characteristic proteins in shRNA and NC cell groups,and to determine the effect of DNMT3B gene silencing on EMT ability of human bladder cancer cells.Results1.RT-PCR showed that compared with NC group,THE mRNA expression of DNMT3B in shRNA group and UMUC3 cells was inhibited to varying degrees(P<0.01),and the silencing efficiency was 56%and 48%,respectively.Thus,bladder cancer cell lines were successfully identified and stably transfected.2.Cell scratch assay showed that the cell migration rate of shRNA group was lower than that of NC group after DNMT3B gene silencing(P<0.05).Transwell cell assay showed that the number of migration and invasion cells in shRNA group after DNMT3B gene silencing was lower than that in NC group(P<0.01).Western Blot analysis showed that EMT characteristic protein expression in human bladder cancer cells decreased after DNMT3B gene silencing(P<0.01).ConclusionRNAi lentiviral vector technology can effectively reduce the mRNA expression of DNMT3B gene in bladder cancer cells,and inhibit the migration,invasion and biological behavior of bladder cancer cells to a certain extent,providing experimental and theoretical basis for the research of human bladder cancer gene therapy.