Effects Of Livin Gene Silencing By ShRNA On Apoptosis And Cycle Of Malignant Melanoma A375 Cells

objective The occurrence of tumor correlates not only with the abnormal proliferation and differentiation of cells,but also with abnormal apoptosis.The family of inhibitor of apoptosis proteins(IAPs) is a class of endogenous cell inhibitory factor,and its over-expression caused by insufficient apoptosis is closely related with the tumor. Therefore,it become research hot spots of cancer gene therapy to look for new ways to treat malignant tumors for IAP target.Livin is a member of the IAP family.Vucic,etc call it the ML-IAP(melanoma IAP),because of the high expression in human melanoma cells. It is specific distributed in a variety of tumor tissues with tumor occurrence,development, prognosis and resistance-associated,will probably become a potential target for tumor treatment.This experiment intends to construct two small interfering RNA eukaryotic expression vector targeting the livin,and observe the influence of apoptosis and cycle of human malignant melanoma A375 cells,in order to explore the gene therapy of maiignant melanoma and provide experimental basis.Methods Human malignant melanoma cell lines A375 were used for the current project.(1) To construct the pGPU6-GFP-livin-shRNA expression vector by gene cloning technology.(2) Expression,intracellular localization and function of the pGPU6-GFP-livin-shRNA in A375 cells were observed by fluorescence microscope after transfection.(3) Access to total RNA of each group,such as Blank group, Lipofectamine group,Negative control group,Experimental group 1 and Experimental group 2,respectively.Expression level of livin mRNA was assayed by real-time fluorescence quantitative PCR technology.(4) Through cells transfection,the apoptosis in A375 cells were analyzed using flow cytometry.(5) The cycle of cells was assessed using Single-PI staining by flow cytometry.Results:(1)The success of gene cloning technology used to build pGPU6-GFP-livin-shRNA expression vector,and liposome-mediated transfection of human malignant melanoma A375 cell lines,green fluorescence was observed under in fluorescence microscopy(2) Expression of livin mRNA of A375 cells after 48h transfected with Experimental group 1 and 2 than the Black control group dropped by 17%and 42%,72h of decreased 71%and 61%.The interferential effect of silent fragments was to strengthen the trend from 48h to 72h.(3) The apoptotic rates of A375 cells transfected with Experimental group1and 2 after 48h which were31.00±2.25%and 34.72±1.29%were higher than those in Blank control group that were 3.21±2.10% (P＜0.05).Liposome group and negative control group(7.67±1.34%and 6.84±1.34%) with no difference compared with blank control group(P＞0.05).The apoptotic rates of A375 cells transfected with Experimental group1and 2 after 72h which were34.90±1.71%and 38.59±1.31%were higher than those in Blank control group that were3.87±3.04%(P＜0.05). Liposome group and negative control group(7.44±1.69%and 7.89±1.44%) with no difference compared with blank control group(P＞0.05).(4) Single-PI staining by flow cytometry showed:Transfected after 48h,shRNA-2 could cause the number of G₀/G₁ phase cells increased,S phase cells decreased,the effect of shRNA-1 was not obvious. Transfected after 72h,shRNA-1 and 2 can cause the number of G₀/G₁ phase cells increased, S phase cells decreased.Conclusion:(1)Design Successfully the oligonucleotide like hairpin structure targeting to total sequence of livin.(2)Construct the shRNA expression vector of pGPU6-GFP-livin-shRNA-1,pGPU6-GFP-livin-shRNA-2,successfully. (3)shRNA-1and2 can effectively reduce expression of livin mRNA in vitro.(4)shRNA-1 and 2 can promote apoptosis of A375 cells.(5) shRNA-1 and 2 enable the cell cycle of human malignant melanoma A375 cells to appear G₀/G₁phase arrest,inhibit cell proliferation.However,the role of cell cycle control point is different in time.(6)The two interferential fragments designed in this experiment are involved in regulation of the expression of livin mRNA in human malignant melanoma A375 cells,can promote the apoptosis of human malignant melanoma A375 cells,and livin gene has the potential to become a new target for gene therapy of malignant melanoma tumor.