Detection Of Inhibition Of Survivin On Induced Apoptosis Of Rat Hsc-6Cell By SiRNA Interference

Objective:To explore the effects of siRNA targeting suvivin on HSC-6and thepossible mechanism.Methods:The experiment was divided into blank group, pGPU6/GFP/Neo-shNCgroup and pGPU6/GFP/Neo-siRNA group.The siRNA was transfected into HSC-T6cells mediated by LipofectamineTM2000for24h, and then the efficiency of transfectionwas observed by fluorescence microscopy. After transfection for48h, the changes ofsurvivin mRNA and protein expression were assessed by reverse transcription-polyme-rase chain reaction(RT-PCR) method and Western-blot,and the form of cells wasobserved by microscopy.. The apoptosis rate of HSC-T6cells was measured by theflow cytometry with PI staining after transfection for48h.The changes of caspase-3protein expression were assessed by western-blot.Results: After transfection for24h，The clear and green fluorescence wasobserved by inversion fluorescence microscope in all siRNA groups，and theefficiency of transfection was attained to75%.After transfection for48h,comparedwith blank group and pGPU6/GFP/Neo-shNC group, the expression of survivinmRNA and protein were obviously weak in pGPU6/GFP/Neo-siRNA group(p<0.05);Compared in the bland group and pGPU6/GFP/Neo-shNC group,theexpression of survivin mRNA and protein was no obviously difference（p>0.05）.Theprominent apoptosis of the pGPU6/GFP/Neo-siRNA group cells was observed bymicroscopy.Compared with blank group and pGPU6/GFP/Neo-shNC group,theapoptosis rate was apparently increased in pGPU6/GFP/Neo-siRNA group(p<0.05);Compared in the blank group, pGPU6/GFP/Neo-shNC group was noobviously difference（p>0.05）. The flow cytometry with PI staining showed that theapoptosis rate was apparently increased in pGPU6/GFP/Neo-siRNA group,comparedwith the blank group,pGPU6/GFP/Neo-shNC group (p<0.05), But there was no obviously difference between blank group and pGPU6/GFP/Neo-shNC group（p>0.05）. Werstern-blot had discovered that caspase-3protein was less in blankgroup and pGPU6/GFP/Neo-shNC group,the expression of caspase-3protein was nosignificant difference（p>0.05）, between them. Then there was significant differencethe expression of caspase-3protein in pGPU6/GFP/Neo-siRNA group compared withother groups (p<0.05).Conclusion:1. siRNA targeting suvivin can inhibit the expression of survivingene and induce apoptosis of HSC-T6cell line；2. siRNA targeting suvivin up-regulated caspase-3and increasedthe apoptotic cells.