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目的:探讨通过RNA干扰技术沉默MACC1基因的表达对卵巢上皮性癌细胞SKOV3顺铂敏感性的影响及其可能机制。方法:前期构建的针对MACC1 mRNA的小发卡状RNA重组表达载体转染卵巢癌SKOV3细胞,获得MACC1基因表达下调的shRNA-MACC1-SKOV3细胞。RT-PCR及Western blot检测MACC1 mRNA和蛋白表达,并检测ERK1/2和p-ERK1/2蛋白含量的变化以及ERK1/2通路抑制剂PD98059对上述蛋白水平的影响。四甲基偶氮唑蓝法(MTT)检测shRNA-MACC1-SKOV3细胞顺铂敏感性变化。同时设置空白组(SKOV3细胞)和转染空质粒组(SKOV3/DDP-EGFP细胞)为对照组。结果:(1)shRNA-MACC1-SKOV3细胞的MACC1 mRNA和蛋白明显降低。(2)shRNA-MACC1-SKOV3细胞p-ERK1/2表达明显降低,在此基础上加入PD98059后p-ERK1/2表达改变更为明显。(3)shRNA-MACC1-SKOV3细胞的顺铂半数抑制浓度(IC50)为(20.420±1.437)μmol/L,明显低于空白组和转染空质粒组[分别为(28.168±0.899)和(27.071±1.229)μmol/L。结论:SKOV3细胞中MACC1基因表达下调导致细胞对顺铂的敏感性增加,可能与下游的ERK1/2通路受到抑制有关。
Objective: To investigate the effect of silencing the expression of MACC1 gene by RNAi on the sensitivity of ovarian epithelial carcinoma cell line SKOV3 to cisplatin and its possible mechanism. Methods: shRNA-MACC1-SKOV3 cells with low expression of MACC1 gene were transfected into ovarian cancer SKOV3 cells. The expression of MACC1 mRNA and protein were detected by RT-PCR and Western blot. The protein levels of ERK1 / 2 and p-ERK1 / 2 and the effect of ERK1 / 2 inhibitor PD98059 on these proteins were detected. Sensitivity changes of cisplatin in shRNA-MACC1-SKOV3 cells were detected by MTT assay. At the same time, the blank group (SKOV3 cells) and the transfected empty plasmid group (SKOV3 / DDP-EGFP cells) were set as the control group. Results: (1) MACC1 mRNA and protein of shRNA-MACC1-SKOV3 cells were significantly decreased. (2) The expression of p-ERK1 / 2 was significantly decreased in shRNA-MACC1-SKOV3 cells, and the expression of p-ERK1 / 2 was more obvious after adding PD98059. (3) The IC50 of shRNA-MACC1-SKOV3 cells was (20.420 ± 1.437) μmol / L, which was significantly lower than that of blank group and transfected empty plasmid group [(28.168 ± 0.899) and ± 1.229) μmol / L. Conclusion: The down-regulation of MACC1 gene expression in SKOV3 cells leads to the increased sensitivity of cells to cisplatin, which may be related to the down-regulation of ERK1 / 2 pathway.