Effects Of RhoC-siRNA Combined With Rapamycin On Proliferation Invasion And Apoptosis Of Hepatocellular Carcinoma Cell BeI7402

Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver. It has highdegree of malignancy with extremely fast growth rate and strong ability of invasion anddistant metastases. About50-100million patients die from liver cancer every year, and itsincidence is still increasing every year. Although the5-year survival of early cancer patientshave increased to75%-80%by surgery,chemoembolization and other means. It is still lackof effective treatment for patients with advanced cancer.RhoC protein, one of the homologous of Rho family,is an important member of RhoGTPases. Abnormal expression of the Rho subfamily proteins may be related with theinvasion and metastasis of malignant tumors.Research shows that over expression of RhoCgene may be related with the invasion, metastasis and apoptosis of liver cancer.mTOR(mammalian target of rapamycin) is the hub of multiple cellular signalingtransduction pathways. Rapamycin (Rapa) is a specific inhibitor of the PI3K/Akt/mTORsignaling pathway, by inhibiting the activity of the mTOR protein to inhibit tumor cellproliferation, invasion, metastasis and apoptosis.The activation of effective molecules MMPs, VEGF, CDK2, P16,Bcl-2and Bax playsan important role of HCC development process. Relative researches show that the overexpression of RhoC or the activation of mTOR can change the expression of MMPs, VEGF,CDK, P16,Bcl-2and Bax.The function of RhoC and mTOR is different while the respective activation induces thesimilar cell biological changes. The activation of RhoC could lead to tumor cellularproliferation,differentiation,invasion,metastasis and apoptosis through MAPK and PI3K/Aktsignal transduction pathway.mTOR is also the downstream target molecule of PI3K/Aktsignal transduction pathway.So we suppose that the expression of MMPs、VEGF、CDK2,Bcl-2and Bax could be regulated by RhoC and mTOR at two different levels and thecombination of RhoC and mTOR suppression may downregulate the expression of MMPs、VEGF、CDK2and Bcl-2gene much deeply and promote the expression of the Bax and P16gene in order to increase the suppressive effect of HCC invasion, metastasis andproliferation. Methods： Bel7402cells were randomly divided into seven groups: blank controlgroup, negative plasmid group, RhoC-siRNA group, RhoC-siRNA combined with Rapagroup, negative plasmid combined with Rapa group, Rapa group and solvent group.Different groups were treated with RhoC-siRNA, Rapa or RhoC-siRNA combined with Rapa.Cell cycle protein related genes ware assessed by semi-quantitative RT-PCR. Metastasis ofcells was determined through MMP-2, MMP-9and VEGF mRNA by RT-PCR. Cellmigration was detected by Transwell. MTT described cell growth curve. Determine the cellapoptosis and apoptosis-related gene by RT-PCR.Results：RhoC silence and Rapa significantly restrained proliferation, metastasis andinvasion of Bel7402, and promote the apoptosis. This effect was more significant in Rapaand RhoC-siRNA combination group. MMP2,MMP9,VEGF,CDK2, P16and Bcl-2mRNAexpression in RhoC-siRNA and Rapa group notably decreased，whereas P16and Bax genesignificantly increased（p<0.05）. The expression of MMP2, MMP9, VEGF,CDK2,and Bcl-2mRNA in RhoC-siRNA and Rapa combination group decreased significantly，while P16andBax gene expression increased significantly（p<0.05）. RhoC-siRNA combined with Rapasignificantly restrained Bel7402migration（p<0.01）and proliferation, meanwhile it promotedBel7402apoptosis（p<0.01）.Conclusions：RhoC gene silencing or Rapa can inhibit Bel7402cell proliferation,invasion and metastasis, meanwhile it promoted Bel7402apoptosis. The combination ofRhoC-siRNA with Rapa can further enhance this effect and better inhibition the developmentof liver tumor.