| Objective:To investigate the effect and mechanism of Ginsenoside Rh2 in suppressing growth and matastasis of hepatoma cells.To provide a theoretical basis for the clinical application of Ginsenoside therapy of hepatocellular carcinoma.Methods:Hepatoma cell lines HepG2,Huh7 and SMMC7721 were treated with Ginsenoside Rh2（20 μg/mL）,after 48 h and then collect cells,the effects of Ginsenoside Rh2 on the expression of miR-29a-5p,miR-26b-3p,miR-224-3p,miR-200b-5p and miR-146a-5p were detected by qRT-PCR.Hepatoma cell lines HepG2 were treated with Ginsenoside.Rh2（20 μg/mL）Id,3d,and 5d,the effect of Ginsenoside Rh2 on the proliferation of HepG2 cells was measured by MTS assay.miR-146a-5p minics,miR-146a-5p inhibitor,negative control were transfected in HepG2 cells respectively,and then cells treated with Ginsenoside Rh2,Cell proliferation was detected by MTS assay,cell migration and invasion were detected by Transwell,cell colony forming ability was detected by colony formation assay,cell apoptosis and cycle were analyzed by flow cytometry,the protein expression levels of MCL 1,MMP 2 and Nfr 2 in cells were measured by Western blot.HepG2 cells infected with miR-146a-5p overexpression and miR-146a-5p inhibitor lentivirus,stable cell lines were constructed.The effects of Ginsenoside Rh2 combined with miR-146a-5p overexpression or miR-146a-5p inhibition on the growth of the tumor were investigated by subcutaneous tumorigenesis in nude mice.The protein expressions levels of MCL 1,MMP 2 and Nrf 2 were detected by Western blot and immunohistochemistry.HepG2 cells were treated with Ginsenoside Rh2（20 μg/mL）,after cultivated 48 h,the cells were collected and the IncRNA chip was used to analyze the effect of ginsenoside Rh2 on the expression of IncRNA in hepatoma cells.Results:Compared with the untreated hepatoma cell group,the relative expression levels of miR-29a-5p and miR-26b-3p in three kinds of hepatoma cells treated with ginsenoside Rh2 were significantly decreased,but the relative expression levels of miR-224-3p,miR-200b-5p and miR-146a-5p were significantly increased,the relative expression level of miR-146a-5p increased largest,the relative expression level in cell lines HepG2,Huh7 and SMMC7721 expression levels were 4.14±0.2,3.12±0.15,3.05±0.02 respectively.The results of MTS experiment showed that Ginsenoside Rh2 could significantly inhibit the proliferation of HepG2 cells.Ginsenoside Rh2 combined with miR-146a-5p overexpression could inhibit the proliferation,migration,invasion and cloning of HepG2 cells,inhibit the cell cycle of HepG2 cells from phase G1 to S phase,inhibit the protein expression of MCL 1,MMP 2,and Nrf 2 in HepG2 cell.The tumorigenesis experiment in nude mice showed that the expression of miR-146a-5p inhibition could significantly promote the growth of tumor,but the combination of Ginsenoside Rh2 and miR-146a-5p overexpression could significantly inhibit the growth of tumor.Western blot and immunohistochemistry results showed that Ginsenoside Rh2 combined with miR-146a-5p overexpression inhibited the expression of protein MCL 1,MMP 2 and Nrf 2 in nude mice.The effects of Ginsenoside Rh2 on the expression of IncRNA and mRNA in HepG2 cells were analyzed by LncRNA microarray,and foud 703 differences expression lncRNAs and 489 differences expression mRNAs,the nunber of upregulation IncRNA was 580,downregulation IncRNA was 123,the nunber of upregulation mRNA was 329,downregulation mRNA was 160.The LncRNA XLOC012708 and LncRNA LOC285547,which have the greatest variation,are likely to participate in Ginsenoside Rh2 inhibition of hepatoma cells growth.Conclusion:In this study,we found that Ginsenoside Rh2 could significantly promote the expression of miR-146a-5p in HepG2 cells,it is revealed that Ginsenoside Rh2 has significant inhibitory effect on the proliferation,migration,invasion and clone formation of HepG2 through miR-146a-5p,provide a theoretical basis and potential therapeutic target for the prevention and treatment of hepatocellular carcinoma metastasis. |
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