沉默MACC1基因对人卵巢癌耐药细胞增殖、凋亡和侵袭的影响

Effects of MACC1 silencing on proliferation,apoptosis,and invasion of human ovarian cancer drug-resistant cell line

目的观察应用基因沉默技术抑制人卵巢癌耐药细胞结肠癌转移相关基因1(MACC1)表达后,细胞增殖、凋亡和侵袭能力的变化及其分子机制。方法将人卵巢癌耐药细胞株SKOV-3/DDP分成空质粒组和转染组,分别转染空质粒p-super-EGFP、重组质粒p-super-EGFP-MACC1 shRNA,另取未经处理的SKOV-3/DDP细胞作为空白对照组。采用RT-PCR方法检测各组MACC1 mRNA表达,Western blotting法检测MACC1蛋白表达,MTT法检测细胞增殖能力,流式细胞术检测细胞凋亡情况,体外黏附试验检测细胞体外黏附力,Transwell小室检测细胞侵袭能力,Western blotting法测定C-met、ERK1/2、p-ERK1/2蛋白表达。结果与空白对照组和空质粒组比较,转染组MACC1 mRNA和蛋白表达减少,细胞48、72 h增殖能力减弱,细胞凋亡率增加,体外黏附和侵袭能力减弱,C-met、pERK1/2蛋白表达增加(P均<0.05),而空白对照组和空质粒组各指标差异无统计学意义(P均>0.05)。结论MACC1基因表达抑制后,SKOV-3/DDP的增殖、黏附和侵袭能力减弱,细胞凋亡增加,这可能与HGF/C-met和ERK通路受抑制有关。

Objective To observe the proliferation,apoptosis,and invasion changes of human ovarian cancer drug-resistant cell line after metastasis-associated in colon cancer-1（ MACC1） silencing by using gene silencing technique and its possible mechanism. Methods The human ovarian cancer drug-resistant SKOV-3/DDP cells were divided into the empty plasmid group and the transfection group,which were transfected with p-super-EGFP and p-super-EGFP-MACC1 shRNA.In addition,the untreated SKOV-3/DDP cells were taken as the control group. The expression of MACC1 mRNA was detected by RT-PCR,Western blotting was used to detect the expression of MACC1 protein,MTT assay was used to detect cell proliferation,flow cytometry was used to detect the apoptosis,the adhesion test was used to detect the adhesion of cells in vitro,Transwell chamber was used to detect cell invasion,and the protein expression of C-met,ERK1/2 and p-ERK1/2was detected by Western blotting. Results Compared with the blank control group and empty plasmid group,the MACC1 mRNA and protein levels were lower,the proliferation ability decreased at 48 and 72 h,apoptosis rate increased,the adhesion and invasion abilities increased,the expression of C-met,p-ERK1/2,MMP-2,MMP-9,and Caspase-3 decreased,and the expression of cleaved caspase-3 protein increased in the transfection group（ all P 〈0. 05）. There was no statistically significant difference between the blank control group and empty plasmid group（ all P〉 0. 05）. Conclusion Silencing MACC1 gene inhibits the proliferation,adhesion,and invasion abilities of SKOV-3/DDP cells,and promotes apoptosis,which may be related to the inhibition of HGF/C-met and ERK1/2 pathway.