MicroRNA介导的RNAi对肿瘤细胞多药耐药基因静默作用的研究
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摘要
目的探讨microRNA(miRNA)介导的RNA干扰(RNAi)对乳腺癌耐药细胞株MCFT/ADR中mdrl基因表达的影响以及细胞多药耐药性(multidrug resistance,MDR)的逆转作用。
方法设计并合成4对编码mdrl pre-miRNA的单链寡核苷酸,退火后将双链寡核苷酸与基于鼠miR-155序列为骨架的polⅡ启动子miRNA表达载体连接构建成重组质粒,将4条重组载体与1条阴性对照载体(mdrl-miRNA-Neg)以脂质体法转染入MCFT/ADR细胞。Real-time RT-PCR检测转染前后mdrl mRNA的表达差异;Western blot分析P-gp蛋白量变化;MTT法测定细胞对阿霉素的药物敏感性。
结果成功构建并转染了4条重组载体,发现其中2条(mdrl-miRNA-1与mdrl-miRNA-2)能够有效抑制mdrl基因的表达。Real-time RT-PCR结果显示转染mdrl-miRNA-1与mdrl-miRNA-2质粒后mdrl mRNA的表达分别下降到原来的42.60%(P＜0.01)和49.23%(P＜0.01)。Western blot检测P-gp蛋白的表达量分别下降到原来的51.61%(P＜0.05)和52.18%(P＜0.05)。转染mdrl-miRNA-1质粒后MCF7/ADR细胞的耐药指数由138倍下降到了55倍,减少至原来的39.85%(P＜0.01),转染mdrl-miRNA-2质粒后的耐药指数由138倍下降到了75倍,减少至原来的54.49%(P＜0.01),均具有高度统计学意义。而mdrl-miRNA-3、mdrl-miRNA-4和mdrl-miRNA-Neg质粒转染后与空白对照组(Blank)相比差异无统计学意义(P＞0.05)。
结论MicroRNA介导的RNAi能够通过降解mRNA而有效的抑制mdrl基因的表达,恢复MCF7/ADR细胞对阿霉素的药物敏感性,从而为肿瘤多药耐药的逆转和提高化疗药物抗肿瘤作用提供了新的治疗途径,也为miRNA在RNAi及基因靶向治疗方面的应用提供了实验依据。
Objective To investigate the effect of microRNA-mediated RNA interference(RNAi) on the expression of mdr1 gene and the reversal of multidrug resistance(MDR) of MCF7/ADR human breast cancer cell line.
Methods Four pairs of single-stranded oligo encoding mdrl pre-miRNA sequence were designed and synthesized.After annealing ds oligo were inserted into polⅡmiRNA expression vector,which is based on the routine miR-155 sequence,to construct recombinant vectors.Then four recombinant vectors and one negative-control vector (mdr1-miRNA-Neg) were transfected into MCF7/ADR cells by liposome.The expression of mdr1 mRNA and P-gp protein were detected by real-time RT-PCR and western blot, respectively.The sensitivity of the cells to adriamycin was detected by MTT assay.
Results Recombinant vectors were successfully constructed and transfected.The results showed that two of four recombinant vectors(mdr1-miRNA-1 and mdrl-miRNA-2) could effectively cleave mdrl mRNA and the expression of P-gp protein was also significantly decreased.The mRNA expression in mdrl-miRNA-1 and mdrl-miRNA-2 transfected MCF7/ADR ceils was significantly reduced to 42.60%(P＜0.01) and 49.23%(P＜0.01),respectively.Western blot analysis demonstrated P-gp expression was significantly reduced to 51.61%(P＜0.05) and 52.18%(P＜0.05),respectively.The resistance index(RI) of MCF7/ADR cells transfected with mdr1-miRNA-1 vector was decreased to 39.85%, from 138-fold to 55-fold(P＜0.01).Mdr1-miRNA-1 vector decreased the resistance index to 54.49%,from 138-fold to 75-fold(P＜0.01).The difference among mdrl-miRNA-3, mdrl-miRNA-4,mdrl-miRNA-Neg and blank control group had no statistical significance(P＞0.05).
Conclusion MicroRNA-mediated RNA interference can effectively inhibit the expression of mdr1 gene by mRNA cleavage and restore the sensitivity of MCF7/ADR cells to adriamycin.These results may provide a new strategy for the reversal of multidrug resistance of tumor cells and lay a foundation for the application of microRNA to RNAi and gene therapy.
方法设计并合成4对编码mdrl pre-miRNA的单链寡核苷酸,退火后将双链寡核苷酸与基于鼠miR-155序列为骨架的polⅡ启动子miRNA表达载体连接构建成重组质粒,将4条重组载体与1条阴性对照载体(mdrl-miRNA-Neg)以脂质体法转染入MCFT/ADR细胞。Real-time RT-PCR检测转染前后mdrl mRNA的表达差异;Western blot分析P-gp蛋白量变化;MTT法测定细胞对阿霉素的药物敏感性。
结果成功构建并转染了4条重组载体,发现其中2条(mdrl-miRNA-1与mdrl-miRNA-2)能够有效抑制mdrl基因的表达。Real-time RT-PCR结果显示转染mdrl-miRNA-1与mdrl-miRNA-2质粒后mdrl mRNA的表达分别下降到原来的42.60%(P＜0.01)和49.23%(P＜0.01)。Western blot检测P-gp蛋白的表达量分别下降到原来的51.61%(P＜0.05)和52.18%(P＜0.05)。转染mdrl-miRNA-1质粒后MCF7/ADR细胞的耐药指数由138倍下降到了55倍,减少至原来的39.85%(P＜0.01),转染mdrl-miRNA-2质粒后的耐药指数由138倍下降到了75倍,减少至原来的54.49%(P＜0.01),均具有高度统计学意义。而mdrl-miRNA-3、mdrl-miRNA-4和mdrl-miRNA-Neg质粒转染后与空白对照组(Blank)相比差异无统计学意义(P＞0.05)。
结论MicroRNA介导的RNAi能够通过降解mRNA而有效的抑制mdrl基因的表达,恢复MCF7/ADR细胞对阿霉素的药物敏感性,从而为肿瘤多药耐药的逆转和提高化疗药物抗肿瘤作用提供了新的治疗途径,也为miRNA在RNAi及基因靶向治疗方面的应用提供了实验依据。
Objective To investigate the effect of microRNA-mediated RNA interference(RNAi) on the expression of mdr1 gene and the reversal of multidrug resistance(MDR) of MCF7/ADR human breast cancer cell line.
Methods Four pairs of single-stranded oligo encoding mdrl pre-miRNA sequence were designed and synthesized.After annealing ds oligo were inserted into polⅡmiRNA expression vector,which is based on the routine miR-155 sequence,to construct recombinant vectors.Then four recombinant vectors and one negative-control vector (mdr1-miRNA-Neg) were transfected into MCF7/ADR cells by liposome.The expression of mdr1 mRNA and P-gp protein were detected by real-time RT-PCR and western blot, respectively.The sensitivity of the cells to adriamycin was detected by MTT assay.
Results Recombinant vectors were successfully constructed and transfected.The results showed that two of four recombinant vectors(mdr1-miRNA-1 and mdrl-miRNA-2) could effectively cleave mdrl mRNA and the expression of P-gp protein was also significantly decreased.The mRNA expression in mdrl-miRNA-1 and mdrl-miRNA-2 transfected MCF7/ADR ceils was significantly reduced to 42.60%(P＜0.01) and 49.23%(P＜0.01),respectively.Western blot analysis demonstrated P-gp expression was significantly reduced to 51.61%(P＜0.05) and 52.18%(P＜0.05),respectively.The resistance index(RI) of MCF7/ADR cells transfected with mdr1-miRNA-1 vector was decreased to 39.85%, from 138-fold to 55-fold(P＜0.01).Mdr1-miRNA-1 vector decreased the resistance index to 54.49%,from 138-fold to 75-fold(P＜0.01).The difference among mdrl-miRNA-3, mdrl-miRNA-4,mdrl-miRNA-Neg and blank control group had no statistical significance(P＞0.05).
Conclusion MicroRNA-mediated RNA interference can effectively inhibit the expression of mdr1 gene by mRNA cleavage and restore the sensitivity of MCF7/ADR cells to adriamycin.These results may provide a new strategy for the reversal of multidrug resistance of tumor cells and lay a foundation for the application of microRNA to RNAi and gene therapy.
引文
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