Experimental Research On The Combined Effects Of RhoC-siRNA And Rapamycin In Hepatocellular Carcinoma Cell Line

The radical resection rate of Hepatocellular carcinoma (HCC) increases greatly than before while the high invasion and metastasis rates after surgical treatment become the bottleneck for HCC. The processes of invasion and metastasis include degradation of extracellular matrix, cellular migration, adhesion, proliferation, angiogenesis and so on.RhoC protein, one of the homologous of Rho family, is an important member of Rho GTPases. It plays a diverse role in cell biology, including cell growth, differentiation and metastasis. mTOR (mammalian target of rapamycin) is the hub of multiple cellular signaling tranduction pathways by enchancing protein transcription through the activation of target molecule p70S6 kinase (S6K 1) and eukaryotic translation initiation factor eIF4E.The activation of effective molecules MMPs, VEGF, CDK, Cyclin, Actin plays an important role of HCC development process. Relative researches show that the overexpression of RhoC or the activation of mTOR enhances the expression of MMP-2,VEGF,CDK,CyclinD1.The function of RhoC and mTOR is different while the respective activation induces the similar cell biological changes. The activation of RhoC could lead to tumor cellular proliferation, differentiation, invasion, metastasis through MAPK and PI3K/Akt signal transduction pathway. mTOR is also the downstream target molecule of PI3K/Akt signal transduction pathway. So we suppose that the expression of MMP-2,VEGF,CDK4,CyclinD1 could be regulated by RhoC and mTOR at two different levels and the combination of RhoC and mTOR suppression may downregulate the expression of MMP-2,VEGF,CDK4,CyclinD1 much deeply in order to increase the suppressive effect of HCC invasion, metastasis and proliferation.The methods are showed as followings:(1) RNA interfering vector targetting RhoC and siRNA-scramble were successfully constructed and transfected into liver cell Bel-7402 with significant inhibiting effeciency confirmed by RT-PCR and Western- blot.(2) The inhibiting effeciency of Bel-7402 by rapamycin at differernt concentration using MTT is conducted in order to choose an effective inhibiting concertration. The inhibiting effeciency in transfected Bel-7402 cells as high as 34.78% while the concentration of rapamycin was 9.14mg/L,and we took the concentration.(3) The cellular proliferation and growth characteristics were conducted using MTT in negative control group, Solvent group, rapamycin group, siRNA-scramble group, siRNA-scramble and rapamycin group, RhoC-SiRNA group, RhoC-SiRNA and rapamycin group. The expression of MMP-2,VEGF,CDK4,Cyclin-D1 in different groups were measured by RT-PCR.The results are showed as followings:The RhoC gene silencing or suppression of mTOR could down-regulate the expression of MMP-2,VEGF,CDK4,Cyclin-D1. The suppressive effect was significantly higher than that in negative control group, Solvent group, siRNA-scramble group; The suppressive effects of MMP-2,VEGF,CDK4,Cyclin-D1 expression in RhoC-SiRNA and rapamycin group were notably lower than that in RhoC-SiRNA group or rapamycin group respectively. The expressions of MMP-2,VEGF,CDK4,Cyclin-D1 were no remarkable differences among the negative control group, Solvent group, siRNA-scramble group.The RhoC gene silencing or suppression of mTOR could suppress the tumor cell proliferation. The Cell Proliferation inhibiting rate was significantly higher than that in negative control group, solvent group, siRNA-scramble group in a time-dependent manner, suggesting that the tumor cell proliferation inhibiting effect be achieved by down-regulating CDK4,Cyclin-D1. The cell proliferation inhibiting rate in RhoC-SiRNA and rapamycin group was significantly higher than that in RhoC-SiRNA group or rapamycin group respectively. The cell proliferation inhibiting rates were no remarkable differences among the negative control group, solvent group, siRNA-scramble group.Conclusion:The RhoC gene silencing or suppression of mTOR could down-regulate the expression of MMP-2,VEGF,CDK4,Cyclin-D1 related to HCC invasion, proliferation and angiogenesis. MTT confirmed that the RhoC gene silencing or suppression of mTOR inhibited the proliferation of tumor cells through down-regulating the expression of CDK4,Cyclin-D1. The combination of RhoC gene silencing and suppression of mTOR could down-regulate the expression of MMP-2,VEGF to attenuate the tumor cell invasion, metastasis and proliferation. The RhoC gene silencing could also reduce the tumor cell migration and adhesion.So the combination of the RhoC gene silencing and suppression of mTOR could notably suppress the HCC invasion and metastasis by degradation of extracellular matrix, cellular migration, adhesion, proliferation, angiogenesis in order to cure the tumor.This research lays a foundation to select RhoC as target molecule in tumor gene therapy both theoretically and experimentally. The combination of the RhoC gene silencing and suppression of mTOR could improve the target treatment effect of HCC, as well as the relationship between RhoC and mTOR.