Ginsenoside Rg3 Inhibits Hepatocellular Carcinoma By ARHGAP9

Hepatocellular carcinoma(HCC)is one of the most aggressive neoplasms characterized by high morbidity and mortality.However,its pathogenesis remains obscure,which hinders the effective diagnosis and treatment of HCC.Recent findings suggested that many members of Rho GAP family,such as p190 Rho GAP,DLC1 and ARHGAP,were highly up-regulated in human hepatocellular carcinoma.Our previous studies proved that the expression of ARHGAP9 was lower in HCC tissues,and the patients with low ARHGAP9 expression had poor prognosis compared to the ones with high ARHGAP9 expression.Moreover,high expression of ARHGAP9 also inhibited the activation of some signal pathways associated with invasion and migration in hepatocellular carcinoma.Therefore,we hypothesized that ARHGAP9 is closely involved in HCC development.Ginsenoside Rg3,a bioactive ginsenoside extracted from Panax ginseng C.A.Meyer,displays significant anti-cancer effects in various tumor types.Shenyi capsule(ginsenoside Rg3 monomer)has been developed into a class I new drug in traditional Chinese medicine for cancer.Many experimental and clinical studies have demonstrated that ginsenoside Rg3 plays a crucial role in the recurrence and metastasis of HCC.However,due to lack of exact molecular mechanisms of action,the application of Rg3 in clinic is limited.Since our previous studies indicated ARHGAP9 plays an important role in the occurrence and development of hepatocellular carcinoma,it is worth further study whether anti-tumor effect of ginsenoside Rg3 in HCC is related to ARHGAP9.Therefore,this study is divided into two parts:(1)To further verify the role of ARHGAP9 in the growth,invasion and migration of hepatocellular carcinoma.(2)To explore whether ginsenoside Rg3 inbits migration and invasion of HCC cells by ARHGAP9.A: Functions of ARHGAP9 in growth and metastasis in hepatocellular carcinoma.Firstly,ARHGAP9 was interferenced and overexpressed in hepatocellular carcinoma cell line by transfection and lentivirus infection.Secondly,the effects of ARHGAP9 on the proliferation,cell cycle,apoptosis,invasion and migration of hepatocellular carcinoma cells were assessed by a series of experiments including CCK-8,flow cytometry and transwell assay.In vivo,Balb/c nude mice(aged 4-6 weeks)were injected intraperitoneally with Hep G2 cells to analyze the effect of ARHGAP9 on tumorigenicity in nude mice.The experimental results are summarized as follows:1.The expression level of ARHGAP9 in six hepatocellular carcinoma cell lines.Among the six HCC cell lines,MHCC-97 L had relatively higher ARHGAP9 level,whereas the expression of ARHGAP9 in the other five cells was relatively low and there is no significant difference among them.Therefore,si RNA or si NC were transfected into MHCC-97 L cells to knock down ARHGAP9.Hep G2 and MHCC-97 H cells were chosen to overexpress ARHGAP9.2.The effect of ARHGAP9 knockdown on proliferation,cell cycle and apoptosis of HCC cells.Silencing of ARHGAP9 significantly promoted the proliferation of MHCC-97 L cells,suppressed cell apoptosis,and increased cell population at S phase.3.The effect of ARHGAP9 overexpression on proliferation,cell cycle and apoptosis of HCC cells.Overexpression of ARHGAP9 significantly inhibited the proliferation of Hep G2 and MHCC-97 H cells,promoted cell apoptosis,and increased cell population at G0/G1 phase.4.The effect of ARHGAP9 on invasion and migration of HCC cells.ARHGAP9 knockdown significantly increased the migration and invasion of MHCC-97 L.On the contrary,ARHGAP9 overexpression significantly suppressed the migration and invasion of Hep G2 and MHCC-97 H cells.5.The effect of ARHGAP9 on the tumor growth rate of nude mice.Compared with the control group,the tumor growth rate of ARHGAP9 overexpression group was significantly decelerated.Since the twenty-fourth day,the tumor volume of ARHGAP9 overexpression group was significantly lower than that in control group(p<0.05).Conclusion: Our results suggest that increased expression of ARHGAP9 could inhibit HCC development.B: Ginsenoside Rg3 inhibits migration and invasion of HCC cells by ARHGAP9.We used CCK-8,Transwell,PCR,Western blot and si RNA interference to observe the effects of different concentrations of 20(R)-gindenoside Rg3 on invasion and migration of hepatocellular carcinoma cells,as well as on the expression level of ARHGAP9 in the cells.The results are as listed as follows:1.The effect of ginsenoside Rg3 on the viability of hepatocellular carcinoma cells.The effect of ginsenoside Rg3 on viability of hepatocellular carcinoma cells was detected by CCK-8 assay.When administered 24 h,the cell viability witnessed no significant difference between administration group(1.25,2.5 and 5 ?g/m L)and the control group.2.Ginsenoside Rg3 inhibited invasion and migration of hepatocellular carcinoma cells.Ginsenoside Rg3 in the concentration of 1.25,2.5 and 5 ?g/m L significantly inhibited the invasion and migration of Hep G2 and MHCC-97 L cell lines.3.Ginsenoside Rg3 increased the expression of ARHGAP9 in hepatocellular carcinoma cells.Compared to the control group,ginsenoside Rg3(2.5 and 5 ?g/m L)significantly increased the expression of ARHGAP9 protein in Hep G2 and MHCC-97 L cell lines.4.ARHGAP9 mediates the anti-migration and anti-invasion effect of Rg3.Silencing of ARHGAP9 significantly attenuates anti-migration and anti-invasion effects of Rg3 in Hep G2 and MHCC-97 L cell lines.Conclusion: Our results suggest that Rg3-induced ARHGAP9 expression contribute to anti-metastasis effects of Rg3 in HCC cells.