Effects Of ShRNA-induced Silence Of TWIST Gene On The Biological Behavior Of Colon Carcinoma Cell Line LoVo

Objective: To investigate the effect of shRNA-induced silence of Twist gene on the biological behavior of colon carcinoma cell line LoVo and to explore the relationship between Twist gene and the growth and metastasis of colorectal carcinoma.Methods: The expression of Twist gene mRNA and protein in human colon carcinoma cell lines(LoVo, SW620, SW480 and SW1116) with different metastatic potentials were detected by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) and Western blot respectively. Taken LoVo cells with relatively high protein expression level of Twist as research object, pGPU6/GFP/Neo-shRNA(Twist) recombinant plasmids were transfected into them via Lipofectamin2000TM, and efficiency of transfection was detected by fluorescence microscopy after 48 hours. The transfected cells were screened by G418 to obtain stably transfected cell strain named Positive group(LoVo-shRNA-Twist cells) and its protein expression of Twist was measured by Western blot compared with Control group(LoVo cells only with Lipofectamine2000TM) and Negative group(LoVo-shRNA-NC cells). Proliferation, apoptosis, migration and invasion of three groups described above were evaluated by MTT assay, FACS method, wound healing assay and Transwell invision assay respectively.Results: Both the expression level of Twist gene mRNA and protein in colon carcinoma cell lines with high metastatic potential(Lovo,SW620) were significantly higher than that in cell lines with low metastatic potential(SW480,SW1116) respectively(P<0.05). Transfection efficiency of the transfected cells was over 70% , and the stably transfected cell strain was screened by G418 at a screening concentration of 500μg/ml.The expression level of Twist protein in Positive group decreased by 73% compared with Control group and Negative group(P<0.05). MTT assay and FACS method showed there were no significant differences among the proliferation and apoptosis of the three groups respectively(P>0.05),but wound healing assay and Transwell invision assay indicated migration and invasion ability of Positive group were remarkably inhibited in contrast with Control group and Negative group respectively (P<0.05).Difference between Control group and Negative group was not statistically significant for this experiment(P>0.05).Conclusion: In conclusion, our results suggest that silencing the expression of Twist weakened migration and invasion ability of LoVo cells in vitro,but that may have no effects on proliferation and apoptosis of LoVo cells. Twist gene is expected to serve as a new useful target for therapy of metastatic colorectal cancer.