Effects Of ShRNA Targeting RhoA On Biological Behaviour Of Human Colorectal Cancer Cell Line LoVo In Vitro And In Vivo

Colorectal cancer is one of the most common malignancies and one important cause of cancer-related morbidity and mortality worldwide. Primary treatment is surgical. However, disease relapse because of undetected metastatic disease occurs in up to 50% of patients. Metastasis formation is an essential aspect of cancer, for which the molecular underpinning has long been subject to debate.Multiple genetic aberrations are required for tumor initiation and progression. Gain-of-function mutations of proto-oncogenes or loss-of-function mutations of tumor suppressor genes play an important role in the invasion and metastasis. Metastasis genes encode homing receptors, their ligands,and extracellular matrix-degrading proteinases, which jointly cause invasion.The Rho small GTPases, a subgroup of the Ras superfamily, Rho GTPases play a major role in a variety of cellular signalling processes involved in cell proliferation, differentiation, cell mobility, apoptosis and membrane trafficking .As one of the Rho small GTPases, GTP-bound active RhoA specifically induces the formation of focal adhesions and the assembly of stress fibers. RhoA was found to be increased in some human cancers and played an important role in the invasion and metastasis of the tumor.But what kind of role RhoA playing was not yet very clear in the development of colorectal cancer. Experimental study on siRNA of RhoA transfecting colorectal cancer cell lines was not yet reported at home and abroad. The first time in the world,we transfected shRNA to human colon cancer cell line LoVo to specifically knockdown endogenous RhoA expression, to analyze the functional role of RhoA in colorectal cancer invasion and metastasis, then to explore a new way of gene therapy in colorectal cancer. The experiment are to be excuted by four sections as follows:Objective:According to RhoA gene sequence of GenBank, to construct small interfering RNA (siRNA) targeting RhoA and its expression vector.Methods:siRNA targeting RhoA was designed, two complementary oligonucleotide strands were synthesized and inserted into pGPU6/GFP/Neo vector,which was then restrictive enzyme digested and sequenced.Results:Two pairs of siRNA targeting RhoA were designed,then were inserted into pGPU6/GFP/Neo vector after annealing. Restrictive enzyme digestion and sequencingconfirmed the vector containing siRNA was what we wanted.Conclusion:The siRNA targeting RhoA and its vector are successfully constructed.Objective:To investigate the inhibitory effect of shRNA targeting RhoA on RhoA gene expression in Human colonic cancer cell Line LoVo transfected by the vector.Methods:The RhoA shRNA was transfected into LoVo cells by the liposome complexmethod and then fluo-rescence photographs were taken。RhoA gene expression was assessed by real-time RT-PCR, and RhoA protein expressionwas tested by Western blot.Results:The expression GFP was successfully observed under fluorescent microscope in Human colonic cancer cell Line LoVo transfected. Compared with untransfected LoVo cells,the expression rates of RhoA mRNA and the expression rates of RhoA protein were(47.66 %),(36.93%),respectively in LoVo.RhoA shRNA-transfected cells,significantly lower than those of control cells (P<0.05).Conclusion:The expression of RhoA gene could be effectively reduced by shRNA in LoVo cells. The RhoA shRNA targeting human RhoA is considered to be possible tool against colorectal cancer. Objective:To study the effect of knockdown of RhoA expression by recombinant plasmid pGPU6/GFP/Neo-RhoA shRNA on the characteristics of human colonic carcinoma cell line LoVo in vitro.Methods:The positive transfected LoVo cell clones were screened with G418. The cell growth curve was obtained by cell counter and the cell doubling time was computed. The cell survival rate was measured by MTT assay.The cell motility and invasion ability in vitro were obtained by cell adhere assay,scratch assay and Transwell experiment.Results:There were no significant difference between untransfected LoVo cells and the LoVo cells transfected with PO in all indicators. Compared with the cells in other two groups, the LoVo cells transfected with P1 grew significantly more slowly (P<0.05), having significantly lower survival rate (P<0.05),also significantly lower in cell adhesion rate,cell motility and cell invasion ability (P<0.05).Conclusion:RhoA shRNA can inhibit the growth,adhesiveness and invasiveness of LoVo cells through intervening the expression of RhoA.Objective:To investigate the effect of silencing RhoA gene with RNA interference (RNAi) technique on colonic cancer LoVo cell proliferation in vivo.Methods:short hairpin RNA (shRNA) eukaryotic expression vector against RhoA gene was constructed, named as plasmid pGPU6/GFP/ Neo-RhoA, and transfected into LoVo cells. Then, the stable transfected and untransfected LoVo cells were injected subcutaneously into nude mice. During a 4-week follow-up period, the sizes and weights of tumors were measured. Moreover, the expression of RhoA,VEGF,CD34 (to observe changes of micro vessel density) and MMP-2 protein protein was observed by immunohistochemical technique.Results:Inhibition of tumor growth was demounstrated after treatment with RhoA shRNA. The volume and weight of tumors were significantly decreased in comparison with those of other control groups(P<0.05). Compared with those of other control groups, The expression of RhoA,VEGF,MVD and MMP-2 in LoVo-P1 group was showed significant decrease.Conclusions:Our results suggest that RhoA gene can act as a crucial therapeutic target for slowing colon cancer growth.