Inhibitory effects of siRNA targeting survivin on the growth of a human melanoma cell line, M14.

Abstract

Objective To study the inhibitory effects of siRNA targeting survivin on the expression of survivin,as well as the apoptosis,proliferation and invasion of a human melanoma cell line,M14.Methods Two siRNAs targeting survivin were designed,chemically synthesized,and used to construct the recombinant plasmids,pRAT-H1.1/neo-survivin-siRNA1 and pRNAT-H1.1/neo-survivin-siRNA2.Then,recombinant plasmids were transfected into M14 cells mediated by Lipofectamine 2000 reagent.Those cells untransfected or transfected with empty vector served as the control.After culture over various periods of time.cells were collected for the detection of mRNA and protein expression of survivin with RT-PCR and Western blotting,respectively,and for the examination of apoptosis and proliferation of M14 cells by flow cytometry and MTT methods,respectively.Also,Transwell assay was performed to detect the invasive capability of M14 cells.Results A statistical decrease in the mRNA and protein expressions of survivin was observed along with an increase in apoptotic rate(x2=31.55,P＜0.01)in M14 cells transfectcd with siRNA-containing plasmid compared with untransfected and empty vector-transfected cells.As MTT assay indicated,on day 4 after the transfcorion,the proliferation of M14 cells was inhibited by(55.4±4.3)%,(34.5±4.3)%and(13.3±4.6)%,with pRNAT-H1.1/neo-survivin-siRNA1,pRNAT-H1.1/neo-survivin-siRNA2 and empty vector,respectively:there was a significant difference among the three groups(P＜0.05).Decreased invasive capability was noticed in M14 cells transfected with siRNA-containing plasmid compared with untransfected cells(all P＜0.05).Conclusions The plasmid containing siRNA against survivin can specifically inhibit the expression of sarvivin,proliferation and invasion of tumor cells,and induce cell apoptosis.The inhibition of survivin expression by siRNA may be a rational approach to the gene therapy for malignant melanoma. Key words: Melanoma; Apoptosis; RNA interference