MiR-1-3p Inhibits Proliferation,Migration And Invasion Of Hepatoma Carcinoma Cells By Targeting CAAP1

Objectives In this study,we investigate the different expression of miR-1-3p between human hepatocellular carcinoma cell line and normal liver cell line and the regulating roles and molecular mechnisms of miR-1-3p on the proliferation,apoptosis,migration and invasion of hepatocellular carcinoma cell lines.This provides a theoretical basis for understanding the early diagnostic and therapeutic target of HCC and new insights into the critical role of miR-1-3p/CAAP1 in HCC development.Methods 1 The over-expression plasmid of miR-1-3p(pcDNA3/pri-miR-1-3p)was constructed and the antisense oligonucleotide of miR-1-3p(miR-1-3p inhibitor)was synthesized,then the human hepatoma cell line HepG2 and SMMC-7721 were transfected with pcDNA3/pri-miR-1-3p plasmid(miR-1-3p group),or pcDNA3 empty vector(pcDNA3 group),or miR-1-3p inhibitor group(miR-1-3p inhibitor group),or out-of-order oligonucleotides(NC inhibitor group).2 Real-time PCR was used to detect the five miRNA’s expressions level in hepatocellular carcinoma HepG2,SMMC-7721,MHCC97-H cells,and Human Normal Hepatocyte LO2 Cells and also to detect the expression levels of miR-1-3p after over-expression or suppression of miR-1-3p in HepG2 and SMMC-7721 cells.3 Data from the Cancer Genome Atlas(TCGA)used to perform differential expression analysis,survival analysis,and correlation analysis.4 The role of different miRNAs on the proliferation of hepatocellular carcinoma cells and the effect of miR-1-3p on the proliferation of hepatocellular carcinoma cells were detected with CCK-8 assay.5 The effect of miR-1-3p on colony formationn of hepatocellular carcinoma cells was examined by cell clonal formation assay.6 Flow cytometry technique was used to detect the effect of miR-1-3p on the apoptosis of hepatocellular carcinoma cells.7 The wound healing test and transwell assay was adopted to analysis the function of miR-1-3p on the migration and invasion of hepatocellular carcinoma cells.8 The candidate target gene of miR-1-3p was predicted with bioinformation and combined with cDNA microarray analysis,then the direct target gene CAAP1 was verified by fluorescent reporter assay.9 The mRNA and protein levels of target gene CAAP1 after over-expression or suppression of miR-1-3p in hepatocellular carcinoma cells were detected with qRT-PCR and Western blot.10 The effect of target gene CAAP1 on proliferation,apoptosis,migration and invasion of hepatocellular carcinoma cells was analyzed by CCK-8 experiment,colony formation experiment,flow cytometry,wound healing test and transwell assay.Finally,rescue experiments were used to confirm that the effects of miR-1-3p on cellular malignant behaviors were directly due to the suppression of CAAP1.Results 1 miR-1-3p expression was aberrantly dowmregulated in HepG2 cells and SMMC-7721 cells compared with human normal hepatocyte LO2 cells,compared with pcDNA3 group,miR-1-3p was significantly up-regulated in miR-1-3p group in hepatocellular carcinoma.miR-1-3p was significantly down-regulated in miR-1-3p inhibitor group in hepatocellular carcinoma compared with NC inhibitor group(P<0.01).2 The expression of miR-1-3p was decreased in patients with hepatocellular carcinoma and correlated with the prognosis of patients(P<0.05).3 CCK-8 assay showed that overexpression of miR-1 in the five miRNAs inhibited the proliferation of HepG2 cells(P<0.01).The cell growth activity of HepG2 and SMMC-7721 after over-expresssion of miR-1-3p were significantly inhibited compared with pcDNA3 group(P< 0.01).The growth activity of HepG2 and SMMC-7721 were promoted compared with NC inhibitor group after suppression of miR-1-3p(P<0.01).4 The colony formation assay showed that the cell colony formation activity of hepatocellular carcinoma cells with over-expresssion of miR-1-3p were significantly inhibited compared with pcDNA3 group after over-expresssion of miR-1-3p(P<0.01).The colony formation activity of hepatocellular carcinoma cells were promoted compared with NC inhibitor group after suppression of miR-1-3p(P<0.01).5 Flow cytometry results showed that after overexpression of miR-1-3p in hepatocellular carcinoma cells,compared with pcDNA3 group,miR-1-3p induced apoptosis;miR-1-3p inhibitor inhibited apoptosis compared with the NC inhibitor group.6 The wound healing test and transwell assay showed that the migration and invasion activity of hepatocellular carcinoma cells with over-expresssion of miR-1-3p were significantly inhibited compared with pcDNA3 group(P<0.01).The migration and invasion activity of HepG2 and SMMC-7721 were promoted compared with NC inhibitor group after suppression of miR-1-3p(P<0.01).7 CAAP1 was identified as the direct target gene of miR-1-3p with bioinformation and fluorescent reporter assay.8 The mRNA and protein levels of CAAP1 were both inhibited in hepatocellular carcinoma cells tranfected with pcDNA3/pri-miR-1-3p,and the mRNA and protein levels of CAAP1 were both upgraded in hepatocellular carcinoma cells tranfected with miR-1-3p inhibitor.9 The expression of CAAP1 in hepatocellular carcinoma was negatively correlated with the expression of miR-1-3p and correlated with the prognosis of patients(P<0.05).10 Functional experiments show that CAAP1 promotes cell proliferation,migration and invasion,and inhibits apoptosis,which is contrary to the effect of miR-1-3p.11 The rescue experiment further confirmed that CAAP1 is a functional target gene of miR-1-3p.Conclusions 1 miR-1-3p inhibits the proliferation and colony formation activities of HepG2 and SMMC-7721 and induces apoptosis.2 miR-1-3p inhibits the migration and invasion of HepG2 and SMMC-7721.3 miR-1-3p negatively regulates CAAP1 mRNA and protein levels,and CAAP1 promotes the proliferation,migration and invasion of hepatocellular carcinoma cells and inhibits the apoptosis of hepatocellular carcinoma cells,indicating that CAAP1 is the direct target gene of miR-1-3p in hepatocellular carcinoma cells and in miR-1-3p may inhibit the proliferation,migration and invasion of hepatocellular carcinoma cells by targeting CAAP1,and induce hepatocellular carcinoma cells apoptosis.