Influence Of Survivin Antisense Oligonucleotide On Survivin MRNA Expression And Chemosensitivity To Paclitaxel In Human Breast Cancer Cells MCF-7

Background & ObjectiveSurvivin,a member of inhibitor of apoptosis protein(IAP)gene family,highly expressed in most common human neoplasms,and is involved in proliferation, apoptosis and chemosensitivity of tumor cells.This study was to investigate the effects of survivin antisense oligonucleotide(ASODN)on the proliferation,apoptosis and Chemosensitivity of human breast cancer cell line MCF-7 to paclitaxel,and explore potential mechanisms.MethodsSurvivin ASODN was transfected into MCF-7 cells mediated by liposome.The expression of survivin mRNA in MCF-7 cells was detected by reverse transcription polymerase chain reaction(RT-PCR).Cell proliferation and chemosensitivity to paclitaxel were detected by MTT assay.Cell cycle and apoptosis were detected by flow cytometry(FCM).Results(1)After 48h from survivin-ASODN exposure,survivin gene expression was efficiently down-regulated.The inhibitory effect were increased with high concentrations of ASODN:A200 group was down-regulated to 86.83%,A400 group was to 76.72%,and A600 group was to 58.49%.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)The proliferation inhibition rate of MCF-7 transfected with ASODN was increased in a dose-and time-independent manner.When treated with 200nmol/L,400nmol/L and 600nmol/L ASODN for 48h,the proliferation inhibition rate of MCF-7 were 22.25%,49.30%and 58.62%.At a concentration of 600nmol/L ASODN,they were 39.60%,61.03%and 70.74%after transfected for 24,48,72h.There were significant difference among different treated groups,P＜0.05.(3)Survivin-ASODN influence MCF-7 cell cycle.The cell cycle of A600 group treated for 24h was arrested at G₂/M phase,and the digital imaging analytical results:the ratio of G₂/M phase of A600 group was(40.58±2.07)%,normal control group was(21.95±0.62)%,lipo group was(20.29±0.78)%,S600 group was (25.37±1.47)%.The apoptotic ratios of MCF-7 cell were increased after treated with 600nmol/L ASODN.The digital imaging analytical results:A600 group was (15.23±1.01)%,normal control group group was(5.09±0.37)%,lipo group was (5.25±0.61)%,S600 group was(6.31±0.72)%.There was significant difference between A600 group and normal control group.P＜0.05.(4)It is clear that the expression of survivin in MCF-7 was changed with different time exposured to paclitaxel.The level of survivin mRNA were(0.66±0.09),(1.26±0.11),(2.34±0.22),(1.01±0.29),(0.32±0.09)in different point-in-time 0,6,12,24,48h,respectively.Paclitaxel could enhance the expression of survivin at an early time,and this effect might play a critical role in the resistence of paclitaxel. There were significant difference between consecutive groups.P＜0.05.(5)The level of survivin mRNA was improved after treated with paclitaxel for 6h in normal group(from0.66±0.09to 1.26±0.11),lipo group(from0.66±0.12to 1.29±0.11) and S600 group(from0.65±0.08to 1.24±0.14).There was significant difference in two groups,P＜0.05.The antisense oligonucletide efficiently down-regulated the induction to(0.51±0.13).There was significant difference between A600 group and normal control group when exposured to paclitaxel for 6h.P＜0.05.(6)The MCF-7 cell transfected with ASODN enhance the chemosensitibity to paclitaxel.The proliferation inhibition rate of A600 group co-cultured with paclitaxel was(82.30±5.26)%,and the rate of normal control group with paclitaxel was (36.93±4.20)%.There was significant difference in two groups.P＜0.05.ConclusionSurvivin antisense oligonucleotide can effectively suppress the expression of survivin mRNA,induce cell cycle arrest at G₂/M phase,inhibit cell proliferation,increase apoptosis;paclitaxel could enhance the expression of survivin at an early time,and this effect might play a critical role in the resistence of paclitaxel:the lower expression of survivin enhance the chemosensitivity to paclitaxel in MCF-7.Survivin target therapy may be used as an important new method in breast cancer treatment.