稳定表达ClC-3对宫颈癌细胞顺铂敏感性的影响及其机制

Effect of stable expression of ClC-3 on sensitivity to cisplatin in cervical cancer cells and its mechanism

目的 :探讨稳定表达电压门控氯通道3(voltage-gated chloride channel 3,ClC-3)的宫颈癌HeLa细胞对顺铂敏感性的变化及其可能的机制。方法 :通过脂质体介导的方法将重组载体质粒pcDNA3.1/ClC-3转染至HeLa细胞中。pcDNA3.1/ClC-3转染后,蛋白质印迹法检测细胞中ClC-3蛋白和P-糖蛋白(P-glycoprotein,P-gp)的表达水平,实时荧光定量PCR法检测细胞中ClC-3和多药耐药基因1(multi-drug resistance gene 1,MDR1)mRNA的表达水平,MTT法检测顺铂对细胞增殖的影响,免疫荧光法检测细胞中ClC-3蛋白和P-gp的表达及定位。结果 :成功获得稳定表达ClC-3的HeLa/ClC-3细胞。HeLa/ClC-3细胞中ClC-3蛋白和P-gp的表达水平以及MDR1和ClC-3 mRNA的表达水平均明显高于转染空载体pcDNA3.1的HeLa/mock细胞,差异有统计学意义(P<0.05)。与转染空载体pcDNA3.1的HeLa/mock细胞比较,顺铂对HeLa/ClC-3细胞增殖的抑制作用明显降低,差异有统计学意义(P<0.05),顺铂对HeLa/ClC-3细胞的半数抑制浓度(half inhititory concentration,IC50)为71μmol/L,明显高于HeLa/mock细胞的32μmol/L(P<0.05)。ClC-3蛋白和P-gp共同定位于HeLa/ClC-3和HeLa/mock细胞的细胞膜。结论 :ClC-3可降低HeLa细胞对顺铂的敏感性,可能与ClC-3上调P-gp的表达水平有关。

Objective： To investigate the stable expression of voltage-gated chloride channel 3（ClC-3） on the sensitivity to cisplatin in cervical cancer HeLa cells, and to explore its possible mechanism. Methods： The recombination vector plasmid pcDNA3.1/ClC-3 was transfected into HeLa cells by liposome-mediated intracellular delivery. After transfection with pcDNA3.1/ClC-3, the expression levels of ClC-3 protein and P-glycoprotein（P-gp） in HeLa cells were determined by Western blotting, and the expression levels of ClC-3 and multi-drug resistance gene 1（MDR1） mRNAs were examined by real-time fluorescence quantitative PCR. MTT assay was used to determine the growth inhibition induced by cisplatin, and then the expressions and location of ClC-3 protein and P-gp were observed by immunofl uorescence. Results： The ClC-3 was stably expressed in HeLa cells, which was named as HeLa/ClC-3. The expression levels of MDR1 and ClC-3 mRNAs as well as ClC-3 protein and P-gp in HeLa/ClC-3 cells were higher than those in HeLa/mock cells transfected with empty vector pcDNA3.1（P 〈 0.05）. As compared with the HeLa/mock cells, the inhibitory effect of cisplatin on HeLa/ClC-3 cells decreased obviously（P 〈 0.05）, and the half inhibitory concentration（IC50） value of cisplatin in HeLa/ClC-3 cells was higher than that in HeLa/mock cells（71 μmol/L vs 32 μmol/L, P 〈 0.05）. The ClC-3 protein and P-gp were co-located at the cell membrane of HeLa/ClC-3 and HeLa/mock cells. Conclusion： ClC-3 can reducethe sensitivity of HeLa cells to cisplatin. This effect is associated with up-regulation of P-gp expression.