MiR-424-5p Affects The Proliferation,Migration And Invasion Of Hepatocellular Carcinoma Cells By Regulating The Expression Of CLDN6

Hepatocellular carcinoma is one of the most common clinical malignancies with a high metastatic potential.Metastases are a major cause of hepatocellular carcinoma recurrence and poor prognosis.CLDN6 is an important member of the CLDNs family.CLDN6 expression is highly tissue specific,with low expression in breast,cervical,prostate and colon cancers but high expression in liver cancer.Previous work by our group has shown that CLDN6 is strongly pronounced human hepatocellular carcinoma tissues and cells,which may promote proliferation,migration and invasiveness of human hepatocellular carcinoma cells.The mechanisms regulating CLDN6 expression in human hepatocellular carcinoma cells remain unclear.MicroRNAs(miRNAs),as short non-coding RNAs,may act differently during tumor development.It has been found that there is a correlation between miRNAs and CLDNs in hepatocellular carcinoma.MiRNAs may target CLDNs to regulate the expression of CLDNs and affects the growth and metastases of hepatocellular carcinoma.Therefore,we speculate that there may exist a miRNAs which can directly target CLDN6 to regulate CLDN6 expression,which in turn affects proliferation,invasion and metastasis of hepatocellular carcinoma cells.Objective:The purpose of this study is to find a miRNA that can target CLDN6 through bioinformatics analysis,and to verify through a series of experiments that this miRNA can directly target CLDN6 and affect the proliferation,migration and invasion of hepatocellular carcinoma cells by regulating expression of CLDN6.Methods:1.Bioinformatics analysis to predict miRNAs targeting gene as CLDN6 in hepatocellular carcinoma cellsFrom four databases of miRNA analyses,miRNAs targeting CLDN6 were predicted in hepatocellular carcinoma and miRNA binding sites to CLDN6 were predicted using Targetscan software.2.Expression of miR-424-5p and CLDN6 in human hepatocellular carcinoma cellsRT-qPCR was performed to detect expression levels of miR-424-5p and CLDN6 in normal liver cells and different kinds of hepatocellular carcinoma cells.3.Confirmation of CLDN6 as a target gene of miR-424-5p in hepatocellular carcinoma cells3.1 Effect of miR-424-5p on the expression profile of CLDN6 in human hepatocellular carcinoma cellsThe protein and mRNA levels of CLDN6 were detected by Western blot and RT-qPCR assays in human hepatocellular carcinoma cells after overexpression of the miR-424-5p.3.2 Dual luciferase reporter analysis confirmed the targeted association of miR-424-5p and CLDN6The binding sites of miR-424-5p and CLDN6 were identified,wild-type and mutant sequences were synthesized,and the synthesized sequences were inserted into the luciferase reporter gene vector,respectively in hepatocellular carcinoma cells.The association between miR-424-5p and CLDN6 was confirmed by measuring luciferase activity in combination with luciferase reporter assay after transfection.4.Effect of miR-424-5p in proliferation,migration and invasion of human hepatocellular carcinoma cells by regulating CLDN6 expression.4.1 Effect of miR-424-5p on the proliferative capacity of human hepatocellular carcinoma cellsOverexpression of miR-424-5p in HepG2 and Huh-7 cells to test the proliferative capacity of human hepatocellular carcinoma cells by CCK-8 assay and clone formation assay.4.2 Effect of miR-424-5p on migration and invasion ability of human hepatocellular carcinoma cellsOverexpression of miR-424-5p in HepG2 and Huh-7 cells to test the migration and invasion ability of human hepatocellular carcinoma cells by wound-healing assay,migrations and invasion assay.4.3 The effect of miR-424-5p on EMT-related protein expression in hepatocellular carcinoma cellsOverexpression of miR-424-5p in HepG2 cells to detect the EMT-related protein expression in human hepatocellular carcinoma by western blot assay4.4 Rescue assayMiR-424-5p mimic/vector and miR-424-5p mimic/CLDN6 experimental groups were set up to perform CCK-8 assay,migrations and invasion assay to test the proliferation,migration and invasion ability of human hepatocellular carcinoma cells.Results:1.Bioinformatics analysis to predict miRNAs targeting CLDN6 in hepatocellular carcinoma cells.Bioinformatic predictive analysis revealed that miR-424-5p can target CLDN6 in human hepatocellular carcinoma cells.2.Expression of miR-424-5p and CLDN6 in human hepatocellular carcinoma cells.RT-qPCR showed that miR-424-5p expression levels were significantly in HepG2,Huh-7,SMMC7721 and SNU-449 cells(PHepG2<0.0001,PSMMC7721<0.01,PHuh-7<0.001,PSNU-449<0.05),with the most significant downregulation in HepG2 and Huh-7.CLDN6 was highly expressed in human hepatoma cells(PHepG2<0.01,PSMMC7721<0.01,PHuh-7<0.001,PSNU-449<0.05),and the difference was significant.3.Confirmation of CLDN6 as a target gene of miR-424-5p in human hepatocellular carcinoma cells.3.1 Effect of miR-424-5p on the expression profile of CLDN6 in human hepatocellular carcinoma cells.RT-qPCR results showed that miR-424-5p mimic reduced the level of CLDN6 mRNA in human hepatocellular carcinoma cells(P<0.01).Western blot results showed the protein expression of CLDN6 was also significantly downregulated(PHepG2<0.01,PHuh-7<0.001)in miR-424-5p mimic group.3.2 Dual luciferase reporter analysis confirmed the targeted association of miR-424-5p and CLDN6.Recombinant plasmids containing wild type(WT)and mutant type(MT)miR-424-5p binding sequences and luciferase of CLDN6 3’UTR were constructed.Compared with the miR-424-5p nc group,the luciferase activity was significantly reduced(PHepG2<0.05,PHuh-7<0.001)in the miR-424-5p mimic+CLDN6 WT 3’UTR group.The results indicated that CLDN6 was the direct target gene of miR-424-5p.4.To investigate the effect of miR-424-5p on proliferation,migration and invasion of human hepatocellular carcinoma cells by regulating CLDN6 expression.4.1 Effect of miR-424-5p on the proliferative capacity of human hepatocellular carcinoma cells.CCK-8 assay showed the proliferative capacity of HepG2 and Huh-7 cells was reduced when overexpressing miR-424-5p.Compared with the control group,the HepG2 cells growth rate after day 3(PDay3<0.05,PDay4<0.01)was significantly decreased,Huh-7 cells growth rate after day 2(PDay2<0.001,PDay3<0.01)was significantly decreased.The results of the clone formation assay showed in HepG2 and Huh-7 cells with overexpressing miR-424-5p,the number of clones formed was very fewer than in the control group(PHepG2<0.01,PHuh-7<0.001).These results suggested that overexpression of miR-424-5p considerably inhibited the proliferative ability of human hepatocellular carcinoma cells.4.2 Effect of miR-424-5p on migration and invasion ability of human hepatocellular carcinoma cells.The results of wound-healing assay showed the migration ability of HepG2 and Huh-7 cells with overexpressing miR-424-5p was significantly decreased compared to the control group,and the difference was significant(P<0.05).The migrations assay showed that the migration rate of HepG2 and Huh-7 cells with overexpressing miR-424-5p was significantly decreased compared with the control group(PHepG2<0.01,PHuh-7<0.0001.The above results indicated that the migratory ability of human hepatocellular carcinoma cells was significantly inhibited after overexpressing miR-424-5p.The invasion assay showed that invasion ability of HepG2 and Huh-7 cells with overexpressing miR-424-5p was significantly weakened compared with the control group(PHepG2<0.001,PHuh-7<0.0001),and the difference was significant.It indicated that the invasion ability of human hepatocellular carcinoma cells was inhibited after overexpressing miR-424-5p.4.3 Effect of miR-424-5p on the EMT-related protein expression in human hepatocellular carcinoma cells.Western blot assay showed that E-cadherin expression was reduced in HepG2 cells compared to the control group(P<0.01),while vimentin and N-cadherin expression was reduced(PN-cadherin<0.05,PVimentin<0.01),and the difference was significant.4.4 Rescue assayThe miR-424-5p mimic/vector and miR-424-5p mimic/CLDN6 experimental groups were set up.The CCK-8 assay showed that simultaneous overexpression of miR-424-5p and CLDN6 in human hepatocellular carcinoma cells can revert the proliferation ability of human hepatocellular carcinoma cells(PDay2<0.001,PDay3<0.01). the migration assay and invasion assay showed that simultaneous overexpression of miR-424-5p and CLDN6 in human hepatocellular carcinoma cells can alter migration and invasion ability of human hepatocellular carcinoma cells(P<0.0001).Conclusion:1.miR-424-5p is lowly expressed in human hepatocellular carcinoma cells.2.miR-424-5p can directly bind to the 3’UTR of CLDN6,which is a direct target gene of miR-424-5p.3.miR-424-5p can affect the proliferation,migration and invasion of human hepatocellular carcinoma cells by regulating the expression of CLDN6.