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

摘要

Objective To observe the biological effect of small interfering (siRNA ) silencing Livin gene expression on human breast cancer MCF‐7 cell and sensitization of siRNA silencing MCF‐7 cell Livin gene on four kinds of chemotherapy drugs .Methods MCF‐7 was transfected by Lipofectamine 2000 systerm .There are 5 groups :control group (no transfection) ,liposomes group ,an‐tisence 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‐7 proliferation .immunohistochemistry was be carried to detect the Livin gene expression of MCF‐7 cells and the change of Livin protein expression by transformation chemo‐therapy drugs .The change of Livin gene expression of breast cancer MCF‐7 cell 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 apop‐tosis with 5‐FU ,EPI ,DXT ,GEM .Results Livin gene was highly expressed in MCF‐7 cell ,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 ,72 h later (P<0 .05) .5‐FU ,EPI ,DXT ,GEM induced the apoptosis of breast cancer MCF‐7 after 48 h and 72 h later ,and it was dose‐dependence .the apoptosis rate of breast cancer MCF‐7 cells 48 h of 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 .%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、72 h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、72 h均能明显的引起细胞的凋亡，且呈剂量依赖性。转染Livin基因48 h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。