The Effect Of Survivin Antisense Oligonucleotide Antisense Transfection To The Growth And Sensitivity To Vinorelbine In MCF-7

Background&ObjectiveSurvivin is a member of inhibitor of apoptosis protein(IAP) gene family,it is highly expressed in most common human neoplasms,and effect the proliferation, apoptosis and chemosensitivity of tumor cells,The study was to explore the effect of survivin antisense oligonucleotide(ASODN) on apoptosis,proliferation and sensitivity to vinorelbine in human breast cancer cells MCF-7.MethodsSurvivin-ASODN was transfected into MCF-7 cells mediated by Lip reagent. The research detected the expression of survivinmRNA by RT-PCR,and survivinprotein expression by Western blot.Apoptosis rate and cell cycle changes in antisense oligonucleotide(ASODN) transfected cells was detected by flow cytometry(FCM).Cell proliferation and chemosensitivity to vinorelbine in MCF-7 was studied by MTT assay.The effect of survivinantisense oligonucleotide(ASODN) in inducing apoptosis of MCF-7 cells was observed under transmission electron microscope(TEM).Results(1)Survivin-ASODN efficiently down-regulated mRNA after 48h of transfection in a concentration-dependent manner.A200 group was down-regulated to 80.24%, A400 group was to 68.52%,and A600 group was to 49.17%.There were significant difference combination ASODN treated and normal control group,P＜0.05.And there were significant difference among different ASODN treated groups,P＜0.05.(2)Survivin-ASODN efficiently down-regulated survivin protein expression in MCF-7 cell lines after 48h of transfection in a concentration-dependent manner.A200 group was down-regulated to 75.14%,A400 group was to 61.09%,and A600 group was to 29.73%.There were significant difference combination ASODN treated and normal control group,P＜0.05.And there were significant difference among different ASODN treated groups,P＜0.05.(3) The proliferation inhibition rate(IR) of MCF-7 cells transfected with ASODN was obviously increased in a dose-and-time dependent manner.Being treated in the groups of A200,A400,A600 for 48h,the proliferation IR of MCF-7 cells were 35.25%,46.41%and 60.36%.For A600 group,they were 29.05%,60.36%and 69.08% after transfected for 24,48,72h.There were significant difference among the differently treated groups,P＜0.05.(4)Survivin-ASODN transfection influence MCF-7 cell cycle.The cyto-cycle of the A600 group when treated for 48h was arrested at G₂/M phase,and the ratio of G₂/M phase of A600-transfected group was(41.63±2.11)%,the normal control group was(20.86±0.72)%,the lipo-transfected group was(23.15±1.07)%,and the S600-transfected group was(24.17±0.96)%.There was significant difference between A600-transfected group and normal control group,P＜0.05.(5)The apoptotic ratios of MCF-7 were increased by Survivin-ASODN transfection.The digital imaging analytical results:A600 group was (18.39±1.06)%,normal control group was(7.05±0.56)%,lipo group was (6.09±0.61)%,S600 group was(6.81±0.74)%.There was significant difference between A600 group and normal control group,P＜0.05.(6)Survivin-ASODN transfection enhanced the chemosensitibity to vinorlbine.The growth rate of ASODN group decreased more and more obviously with the increased concentration of ASODN.The 50%inhibitory concentration(IC₅₀) of vinorelbine was significantly lower for A600 group than for control cells.The IC50 of A200,A400 and A600 were(5.1±0.52)μg/mL,(4.6±0.57)μg/mL and(3.7±0.42)μg/mL;while the IC₅₀ of S600 group,Lip group and normal group were(6.6±0.74)μg/mL,(6.8±0.81)μg/mL and(7.1±0.68)μg/mL.Being compared,the IC₅₀ of ASODN groups were decreased obviously,and there were significant difference among different treated groups, P＜0.05.(7)Terminal apoptosis changes were observed under transmission electron microscope in most of the cells in A600 groups.The nuclei became pycnotic and the cell membrane sprouted and formed vacuoles and apoptotic bodies.While the phenomenon was not appear in the compared groups.ConclusionSurvivin antisense oligonucleotide(ASODN) effectively suppresses the expression of survivinmRNA and protein,induce cell cycle arrested at G₂/M phase,increases apoptosis,inhibits cell proliferation;and the lower expression of survivinenhanced the chemosensitivity of MCF-7 to vinorelbine.Then,survivin targeting therapy may be used as an important new method in breast cancer chemotherapy.