The Study Of MiR-449a Targets Notch1 Pathway Influencing Hepatoma Cells Invasion And Migration

Objective: To study the molecular mechanism of miR-449 a and NOTCH1 in hepatocellular carcinoma and its adjacent tissues,and to further study the molecular mechanism of miR-449 a affecting hepatocellular carcinoma cell migration and invasion through targeting NOTCH1 pathway in hepatocellular carcinoma.To provide the basis of early diagnosis,effective treatment and prognosis assessment for hepatocellular carcinoma.Methods:(1)The collected 55 pairs of hepatocellular carcinoma and para-cancerous tissue samples were extracted from tissue total RNA,real-time quantitative PCR detection miRNA-449 a gene and NOTCH1 gene expression levels.(2)The hepatoma SMMC7721,HL-7702,QGY-7703,BEL-7402 and Hep G2 cell lines were cultured and the expression levels of miRNA-449 a and NOTCH1 were detected by real-time quantitative PCR.(3)Luciferase reporter assay confirmed that miR-449 a has a specific binding site on the NOTCH1 gene.(4)The SMMC7721 cells were transfected with miR-449 a mimics and miR-449 a mimics NC respectively.QGY-7703 cells were transfected with miR-449 a inhibitor and miR-449 a inhibitor NC respectively.The transfected cells were transfected 24 h,48h and 72 h respectively,and select the best transfection time,and do Western blot experiments.(5)The SMMC-7721 cell line with the lowest expression of miRNA-449 a and the QGY-7703 with the highest expression of miRNA-449 a were selected for scratch test.(6)The SMMC-7721 cell line with the lowest miRNA-449 a expression level and the QGY-7703 with the highest miRNA-449 a level were selected for Transwell experiments.(7)The SMMC-7721 cell line was transfected with miRNA-449 a mimics,miRNA-449 a mimics NC,and miRNA-449 a mimics + NOTCH1,respectively.QGY-7703 cell line,repeated scratch test and Transwell test,and do the recovery experiment.Results:(1)The results of real-time quantitative PCR showed that the expression of miRNA-449 a in hepatocellular carcinoma was significantly lower than that in normal liver tissue.The expression of NOTCH1 gene in hepatocellular carcinoma was significantly higher than that in normal liver tissues(P <0.001).(2)Cytology showed that miR-449 a was overexpressed in SMMC7721,QGY7703,BEL7402 and Hep G2 cell lines,which was significantly lower than that in HL7702 cells.The expression of NOTCH1 gene was highly expressed in SSMMC7721,QGY7703,BEL7402 and Hep G2 cell lines,Significantly higher than that of HL7702 cells.(3)Dual luciferase reporter assay showed that miR-449 a had a direct target on the NOTCH1 gene(P <0.01).(4)Scratch experiments showed that miR-449 a could inhibit the hepatocellular carcinoma cell migration in vitro(P <0.01).(5)Transwell experiments showed that miR-449 a could inhibit the invasion of hepatocellular carcinoma in vitro(P <0.01).(6)Response experiments showed that NOTCH1 can reverse the effect of miR-449 a on hepatocellular carcinoma cell migration and invasion.Conclusions: The results showed that the expression of miR-449 a in hepatocellular carcinoma was significantly lower than that in normal liver tissues.The expression of NOTCH1 protein in hepatocellular carcinoma was significantly higher than that in normal liver tissue;miRNA-449 a can inhibit the migration and invasion of liver cancer cells;NOTCH1 gene can promote the migration and invasion of liver cancer cells;miRNA-449 a inhibits hepatocellular carcinoma cell migration and invasion by targeting NOTCH1 pathway in hepatocellular carcinoma.