生存素反义寡核苷酸在胃癌化疗中对表阿霉素的增敏性及其信号转导途径研究

Enhancement of cytotoxic effect of epirubicin on gastric carcinoma cell line SGC-7901 by survivin antisense RNA and signaling pathway

目的:通过构建survivin反义寡核苷酸,研究其在胃癌化疗中对表阿霉素(Epirubicin,EPI)的增敏作用及作用机制。方法:构建survivin反义寡核苷酸链(Antiscnse RNA,ASODN)及正义寡核苷酸对照链(SODN),脂质体法转染胃癌SGC-7901细胞,MTT法检测转染组细胞抑制率并筛选出适宜浓度,不同浓度表阿霉素处理胃癌SGC-7901细胞,MTT法检查表阿霉素对其生长抑制作用并筛选出4组浓度做为待测浓度,将2种因素联合作用于SGC-7901细胞,Western-blot法检查survivin蛋白表达情况,MTT法检查表阿霉素对其抑制率,流式细胞仪检查细胞周期,Annexin V-FITC法检查细胞凋亡情况,RT-PCR法检查bcl-2、SMac mRNA表达情况。结果:经200nmol/L survivin反义寡核苷酸处理后的SGC-7901细胞对表阿霉素的敏感性明显增加:MTT法示转染后细胞经表阿霉素作用24h后,抑制率较单独使用表阿霉素的未转染组明显提高(P<0.05,q>1.15),流式细胞仪检查示联合作用组使细胞G2/M期细胞数较单独使用表阿霉素组明显增多:Western-blot检查示转染后survivin蛋白表达水平显著降低,Annexin V-FITC检查示转染组细胞凋亡明显增加,RT-PCR检查示:SMac mRNA在联合作用组较空白对照组表达上调。结论:Survivin反义寡核苷酸能够增强SGC-7901细胞对表阿霉素的敏感性,其作用可能是通过上调SMac的表达实现的。

Objective： To investigate whether interfering survivin has the potential to sensitize stomach cancer cells to Epirubicin. Methods： By the method of liposome transfection, survivin antisense oligodeoxynucleotide was transfected into human stomach cancer SGC-7901 cell line. Treat cells with different concentrations of survivin ASODN and Epirubicin respectively to select the best combination trough assessing the growth of cells by MTT assay. Cells were randomly divided into control group,ASODN group, Epirubicin and combination group. Western blot analysis was used to check the survivin protein expression;inhibitive rate were detected by MTT assay; Flow cytometric analysis was used to detect the cycle status of SGC-7901 cells. The apoptosis was screened according to the result of Annexin V-FITC. Reverse transcription polymerase chain reaction（ RT-PCR ）were applied to detect the expression of bcl-2 ,SMac mRNA. Results： MTF assay showed the proliferation of SGC-7901 cells was retarded obviously contrasting to control group. The level of survivin protein expression in ASODN group are lower than control group according to Western blot analysis. Annexin V-FITC show that the apoptosis rate of ASODN group cells increases . The level of SMac mRNA expression in ASODN group are higher than control group according to RT-PCR analysis. Conclusion： Survivin siRNA can sensitize stomach cancer cells to Epirubicin. Up-regulating the SMac expression might be one of mechanisms.