靶向人多药耐药基因 mrp1特异性小分子干扰 RNA 有效序列筛选

摘要

Objective:To screen effective sequences of small interfering RNA targeting human multidrug associat-ed - protein gene(mrp1). Methods:Four siRNAs(mrp1 - si251,mrp1 - si480,mrp1 - si795,mrp1 - si1016)targe-ting mrp1 genes were designed and synthesized by in vitro transcription. The siRNA duplexes were used to transfected into the human hepatocellular carcinoma HepG2 / mrp1 cells,which transfected with human mrp1 gene and obtained MDR phenomenon. The expression level of mrp1 mRNA was detected by RT - PCR. The multidru resistance - associ-ated protein and accumulation of intracellular daunorubicin(DNR)were examined by flow cytometry,respectively. The cell sensitivity to adriamycin(ADM)was demonstrated by MTT. Results:The HepG2 / mrp1 cells treated with 4 siR-NAs led to reversal effectively on multidrug resistance to different extents. Among the HepG2 / mrp1 cells treated by siRNAs for 72 h,the expression level of mrp1 mRNA in cells of mrp1 - si1016 or mrp1 - si795 groups[(85. 54 ± 1. 04)% or(86. 36 ± 2. 26)% ]was more decreased than that in cells of mrp1 - si251 or mrp1 - si480 groups(P ﹤ 0. 05). The accumulation of DNR in cells of mrp1 - si795 group was the most. In cells of mrp1 - si1016 group,more,in cells of mrp1 - si480 group,lower,and in cells of mrp1 - si251 group,the lowest(P ﹤ 0. 05). The rel-ative reversal efficiency of cells of mrp1 - si1016 and mrp1 - si795 groups to ADR was higher than in the cells of mrp1 - si251 and mrp1 - si480 groups significantly(P ﹤ 0. 05). The expression level of multidrug resistance - associ-ated protein in cells of mrp1 - si1016 and mrp1 - si795 groups was lowest among the HepG2 / mrp1 cells treated by siRNAs for 72h. Conclusion:The mrp1 - si795 with most,mrp1 - si1016 with more,mrp1 - si480 with less and mrp1- si251 with least reversal effects on mrp1 gene mediated multidrug resistance were found in the human hepatocellular carcinoma HepG2 / mrp1 cells.%目的：筛选靶向人多药耐药相关蛋白基因（mrp1）特异性小分子干扰 RNA（siRNA）的有效序列。方法：设计并体外转录合成靶向 mrp1的4条 siRNA（mrp1- si251，mrp1- si480，mrp1- si795，mrp1- si1016和空白对照 si -阴性），转染转基因单因素肝癌多药耐药细胞 HepG2/ mrp1。用 RT - PCR 检测 mrp1 mRNA 表达，流式细胞仪检测多药耐药相关蛋白表达、细胞内柔红霉素（DNR）蓄积，四甲基偶氮唑蓝（MTT）法检测细胞对阿霉素（ADM）的敏感性。结果：4条 siRNA 均能不同程度逆转 HepG2/ mrp1细胞由 mrp1介导的多药耐药。转染72h 后，mrp1- si795组和 mrp1- si1016组的 mrp1 mRNA 表达水平分别分别下调了（86.36±2.26）％和（85.54±1.04）％，较 mrp1- si251组和 mrp1-si480组明显下降（P ﹤0.05）；细胞内柔红霉素蓄积由多到少依次为 mrp1- si795组最多，mrp1- si1016组次之，mrp1- si480组较少，mrp1- si251组最少（P ﹤0.05）；mrp1-si795组对 ADR 耐药的相对逆转率（86.36％）最高，mrp1- si1016组（85.54％）次之，较 mrp1- si251组（60.93％）和 mrp1- si480组（70.29％）有明显差异（P ﹤0.05）；mrp1- si1016组和 mrp1- si795组多药耐药相关蛋白表达明显下调。结论：实验设计的靶向 mrp1 mRNA 的 siRNA 序列能够不同程度逆转多药耐药相关蛋白介导的人肝癌耐药细胞 HepG2/ mrp1的多药耐药性，mrp1- si1016和 mrp1- si795效果最好，mrp1- si480较差，mrp1- si251最差。mrp1- si1016和 mrp1- si795可以作为进一步实验的靶序列。