siRNA沉默Livin基因对乳腺癌MCF-7细胞化学治疗增敏作用的研究

Chemosensitization effect of siRNA silence Livin gene on breast cancer MCF-7 cell

目的观察小干扰RNA(siRNA)沉默Livin基因对人乳腺癌细胞MCF-7细胞生物学影响及siRNA沉默MCF-7细胞Livin基因对4种化学治疗药物的增敏作用。方法 Lipofectamine 2000脂质体转染法转染MCF-7。分组:空白组(无转染)、脂质体组、反义组、错义组、联合组。MTT法测定氟尿嘧啶(5-FU)、表柔比星(EPI)、多西紫杉醇(DXT)、吉西他滨(GEM)单药(单药组)对乳腺癌细胞MCF-7增殖的抑制作用。免疫组织化学检测Livin蛋白在MCF-7细胞中的表达及转染联合化学治疗药物对Livin蛋白的表达变化。实时荧光定量PCR(RT-PCR)检测转染Livin siRNA后乳腺癌MCF-7细胞的Livin基因表达变化。同时利用Annexin-V检测乳腺癌细胞的凋亡及siRNA Livin联合5-FU、EPI、DXT、GEM诱导乳腺癌细胞凋亡的影响。结果 Livin在MCF-7细胞中高表达,4种化学治疗药物对Livin表达无明显影响,转染Livin基因48、72h后联合4种药物后能明显下调Livin蛋白表达,差异有统计学意义(P<0.05)。5-FU、EPI、DXT、GEM治疗乳腺癌MCF-7细胞48、72h均能明显的引起细胞的凋亡,且呈剂量依赖性。转染Livin基因48h联合4种药物后乳腺癌MCF-7细胞的凋亡率明显高于单药处理的细胞,差异有统计学意义(P<0.01)。结论 siRNA沉默Livin基因能促进由化学治疗药物5-FU、EPI、DXT、GEM引起的细胞凋亡,具有化学治疗增敏作用。

Objective To observe the biological effect of small interfering（siRNA）silencing Livin gene expression on human breast cancer MCF-7cell and sensitization of siRNA silencing MCF-7cell Livin gene on four kinds of chemotherapy drugs.Methods MCF-7was transfected by Lipofectamine 2000 systerm.There are 5groups：control group（no transfection）,liposomes group,antisence group,missense group and combination group.MTT assay was be used to detect the inhibition effect of 5-fluorouracil（5-FU）,epirubicin（EPI）,docetaxel（DXT）,gemcitabine（GEM）on breast cancer cell MCF-7proliferation.immunohistochemistry was be carried to detect the Livin gene expression of MCF-7cells and the change of Livin protein expression by transformation chemotherapy drugs.The change of Livin gene expression of breast cancer MCF-7cell which was transformation Livin siRNA by RT-PCR examination.At the same time,Annexin-V was used to detect breast cancer apoptosis and siRNA Livin induced breast cancer apoptosis with 5-FU,EPI,DXT,GEM.Results Livin gene was highly expressed in MCF-7cell,there were no obvious differences on Livin expression of four kinds of chemotherapy drugs,the four drugs reduced the expression of Livin gene protein significantly after Livin gene was transfected 48,72hlater（P〈0.05）.5-FU,EPI,DXT,GEM induced the apoptosis of breast cancer MCF-7after 48 h and 72 hlater,and it was dose-dependence.the apoptosis rate of breast cancer MCF-7cells 48 hof combination group was higher than that of the group which use only chemotherapy drugs significantly（P〈0.01）.Conclusion siRNA silencing Livin gene could accelerate 5-FU,EPI,DXT,GEM induced cell apoptosis and it possess chemotherapy sensitization.