Effect Of MiR-29a Targeting SENP1 On Proliferation And Invasion Of Hepatocellular Carcinoma Cells

Background and purpose Hepatocellular carcinoma(HCC)is a common primary malignant tumor of the liver,and its pathogenesis has not yet been fully elucidated.The small ubiquition related modifier(SUMO)is a ubiquitous protein post-translational modification method in cells.SENPs(SUMO-specific proteases,SENPs)participate in the regulation of SUMOylation by removing the SUMO protein from the target protein,and participate in transcriptional regulation and signal transduction of genes.It was found that SENP1(SUMO-specific protease1,SENP1)can maintain the stem cell characteristics of liver cancer stem cells and promote tumor formation;however,its upstream regulatory factors are still unclear.Numerous studies have shown that a variety of small molecule non-coding RNAs(miRNAs)participate in the development of HCC by negatively regulating gene expression by interacting with the 3'UTR region of the target mRNA.Studies have shown that miR-29 a can play a role as a tumor suppressor gene and is lowly expressed in a variety of malignant tumors including HCC.We hypothesized that miR-29 a may regulate the expression of SENP1 by some mechanism to inhibit the malignant biological behavior of hepatoma cells.In this study,we first studied the expression levels of SENP1 and miR-29 a in HCC,and further explored the effects of both on the proliferation and invasion of hepatoma cells,and verified that SENP1 is the target protein of miR-29 a by biological experiments.Through this study,we can further understand the molecular pathogenesis of liver cancer and provide new ideas for the diagnosis and treatment of liver cancer.Method 1.The expression of miR-29 a and SENP1 in HCC was detected by immunohistochemistry,Western blot and real-time fluorescent quantitative PCR.2.The SENP1-RNAi lentiviral vector and miR-29 a mimics were constructed and transfected into MHCC97 H and SMMC7721 cells,respectively.The expression of miR-29 a and SENP1 was further detected by WB and qPCR,respectively.3.Edu,cell cycle and colony formation assay were used to detect the changes of proliferation of MHCC97 H and SMMC7721 cells by transfected SENP1 and miR-29 a.The expression of cyclinA2 and cyclinE1 was detected by Western blotting.4.Flow cytometry was used to detect the changes of apoptosis ability of SENP1 and miR-29 a on hepatoma cells after transfection.5.Transwell and scratch assays were used to detect the changes of invasion and migration of hepatoma cells by transfected SENP1 and miR-29 a.The expression of MMP2 and MMP9 protein was detected by Western blot.6.The miRNA database predicts the target gene and further validates it by dual luciferase reporter assay and Western blot.Result 1.Immunohistochemistry showed that SENP1 was highly expressed in HCC tissues and low in non-cancerous tissues.Compared with human immortalized normal liver cells L02,SENP1 was highly expressed in HCC cells,while miR-29 a was lowly expressed.2.The SENP1-RNAi lentivirus stably transformed strain was successfully constructed.WB and qPCR showed that the expression level of SENP1 protein and mRNA was down-regulated in hepatoma cells after transfection of lentivirus.3.The hepatocarcinoma cells with up-regulated expression of miR-29 a were successfully constructed.qPCR showed that the expression of miR-29 a was up-regulated in hepatoma cells transfected with miR-29 a mimics,but the level of SENP1 mRNA was not significantly changed.WB showed that the level of SENP1 protein was down-regulated after transfection.4.Edu proliferation assay and cell cycle assay showed that after knockdown of SENP1 or up-regulation of miR-29 a expression,hepatoma cells MHCC97 H and SMMC7721 were arrested in S phase,and cell proliferation was weakened.Cell clone formation experiments showed that after knocking down the expression of SENP1,The cloning ability of liver cancer cells MHCC97 H and SMMC7721 was weakened;at the same time,the expression of related cell cycle regulatory proteins cyclinA2 and cyclinE1 was down-regulated.5.Apoptosis experiments showed that apoptosis of liver cancer cells MHCC97 H and SMMC7721 increased after knockdown of SENP1 or up-regulation of miR-29 a expression.6.Transwell experiments and scratch experiments showed that knocking down SENP1 or up-regulating the expression of miR-29 a,the invasion and migration ability of liver cancer cells was weakened;while the protein levels of MMP2 and MMP9 were low.7.TargetScan,PicTar and microRNA.org target gene prediction sites have binding sites for miR-29 a and the 3' terminal untranslated region(3'UTR)of SENP1.The dual luciferase reporter assay further shows that miR-29 a mimics can The activity of the wild type SENP1 reporter gene luciferase was inhibited,but the inhibition of the mutant was not significant.Conclusions 1.SENP1 is highly expressed in HCC tissues and cells,and miR-29 a is under expressed in HCC cells.2.Down-regulation of the expression of SENP1 can inhibit the proliferation and invasion of liver cancer cells MHCC97 H and SMMC7721,and promote their apoptosis,and at the same time cause the down-regulation of MMP9,MMP2,cyclinE1 and cyclinA2.3.miR-29 a can specifically regulate the expression of SENP1 in hepatoma cells and inhibit the proliferation,migration and invasion of hepatoma cells.