MicroRNA-21 Promotes Cell Proliferation In Human Hepatocellular Carcinoma Partly By Targeting HEPN1

Objective: To dicuss the interaction of HEPN1 gene(hepatocellular carcinoma, down-regulated 1) with the miR-21, and the action mechanism of HEPN1 gene in the development of HCC.Methods:(1) Using bioinformatics tools to initially identify the potential targeted relationship between the HEPN1 gene and the miR-21.(2) Using the tecniques of Realtime PCR and Western blot to detect the expression quantity of HEPN1 gene and miR-21 from 72 pairs of HCC tissue and their para-carcinoma tissue, then to analyze their relevance of expression level.(3) The reporter vectors including the pGL3-HEPN1-WT with target site sequence and the pGL3-HEPN1-MT without target site sequence were constructed, then together with the miR-21 mimic(0-100 nM) introduced into the HCC line HepG2 and Hep3 B respectively. To confirm the targeted relationship between HEPN1 gene and miR-21 by dual luciferase reporter gene assay.(4) Introducing miR-21 inhibitor(0-200 nM) of different concentrations into the HCC line HepG2 and Hep3 B to observe the expression level of HEPN1 gene, mi R-21 and the cell proliferation changes. The miR-21 inhibitor and siRNA-HEPN1 were jointly introduced into hepatoma cell line to verify whether the HCC cell proliferation changes were mediated by the HEPN1.Results:(1) The bioinformatics tools initially identified the potencial targrted relationship between HEPN1 gene and miR-21 existed.(2) RT-PCR test results showed that the expression level of HEPN1 in adjacent cancerous tissues was 33.5 times higher than in the hepatocellular carcinoma(P<0.001), while the expression level of miR-21 in the adjacent cancerous tissues was 3.21 times lower than in the hepatocellular carcinoma(P<0.001), and their expression level has significantly negative correlation(R2=0.442,P<0.0001). The HEPN1 protein expression level was consistent with the results of RT-PCR.(3) The dual luciferase reporter gene assay test results showed that the fluorescence intensity of pGL3-HEPN1-WT recombinant vector were significantly lower than the fluorescence intensity of p GL3-HEPN1-MT recombinant vector after introducing the different concentrations of miR-21 mimic. At the same time, the higher of the mi R-21 mimic concentration, the markedly lower of the fluorescence intensity of pGL3-HEPN1-WT and pGL3-HEPN1-MT recombinant vectors, and they both present a tendency of miR-21 mimic concentration dependence. But the slope analysis found a significant difference in the degree of fluorescence decrease between two of them(Slope difference<0.001).(4) After different concentrations of miR-21 inhibitor were introduced into two kinds of hepatoma cell lines, their miR-21 expression levels were significantly decreased(P<0.05), while HEPN1 gene in the m RNA and the protein levels experienced a significant increase(P<0.05). Test results of the MTT showed that with the concentration of miR-21 inhibitors increased, the hepatocellular carcinoma cell proliferation was significantly inhibited. And this inhibitory effect can be partly reversed by siRNA-HEPN1.Conclusion:(1) The HEPN1 gene may be one of the potential target genes of MiR-21.(2) The miR-21 may promote the hepatocellular carcinoma cell proliferation, thus participate the occurrence of HCC.