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目的研究FANCF基因沉默对乳腺癌MCF-7细胞生物学特性的影响,并检测相关指标的变化,探讨FANCF基因是否参与乳腺癌的发生、发展及耐药性的形成。方法构建FANCF-shRNA质粒,转染FANCF-shRNA使乳腺癌MCF-7细胞FANCF基因沉默;MTT法检测FANCF基因沉默对MCF-7细胞增殖的影响;流式细胞仪PI单染法及AnnexinV-FITC/PI双染法检测FANCF基因沉默对MCF-7细胞周期及凋亡的影响;RT-PCR检测FANCF基因沉默前后耐药相关基因mRNA表达水平的变化。结果成功构建FANCF-shRNA质粒,RT-PCR验证转染FANCF-shRNA后48h出现FANCF基因沉默;与阴性对照组相比,FANCF基因沉默后,MCF-7细胞增殖明显受到抑制,转染FANCF-shRNA后48h和72h的抑制率分别为12.65%±1.24%和29.64%±0.87%(P<0.05);S期MCF-7细胞比率增加(33.38%±0.68%,P<0.05),细胞周期阻滞在S期;转染FANCF-shRNA后MCF-7细胞凋亡率明显增加,转染后48h和72h的凋亡率分别为28.95%±1.81%和49.00%±2.32%(P<0.05);P-gp、BCRPmRNA表达水平降低,MRP、LRPmRNA表达水平无明显变化(P>0.05)。结论FANCF基因沉默可抑制人乳腺癌MCF-7细胞增殖,促进细胞凋亡,并使细胞周期阻滞于S期;并可抑制BCRP等耐药相关基因的mRNA表达,参与乳腺癌细胞耐药性的调控。
Objective To investigate the effect of FANCF gene silencing on the biological characteristics of breast cancer MCF-7 cells and to detect the changes of related indicators to investigate whether FANCF gene is involved in the occurrence, development and drug resistance of breast cancer. Methods The FANCF-shRNA plasmid was constructed and transfected into FANCF-shRNA to silence the FANCF gene in breast cancer MCF-7 cells. MTT assay was used to detect the effect of FANCF gene silencing on the proliferation of MCF-7 cells. Flow cytometry PI staining and Annexin V-FITC / PI double staining was used to detect the effect of FANCF gene silencing on the cell cycle and apoptosis of MCF-7 cells. RT-PCR was used to detect the expression of drug resistance-related genes before and after FANCF gene silencing. Results FANCF-shRNA plasmids were successfully constructed and FANCF gene silencing was observed 48 h after transfection with FANCF-shRNA by RT-PCR. Compared with the negative control group, the proliferation of MCF-7 cells was significantly inhibited by FANCF gene silencing, The inhibitory rates at 48h and 72h were 12.65% ± 1.24% and 29.64% ± 0.87%, respectively (P <0.05). The rate of MCF-7 cells in S phase increased by 33.38% ± 0.68% In S phase, the apoptosis rate of MCF-7 cells was significantly increased after transfection with FANCF-shRNA, the apoptotic rate was 28.95% ± 1.81% and 49.00% ± 2.32% (P <0.05) at 48h and 72h respectively after transfection; P -gp, BCRP mRNA expression levels decreased, MRP, LRP mRNA expression levels did not change significantly (P> 0.05). Conclusion FANCF gene silencing can inhibit the proliferation of human breast cancer MCF-7 cells, promote cell apoptosis and arrest the cell cycle in S phase. It can also inhibit the mRNA expression of BCRP and other drug resistance-related genes, and participate in the drug resistance of breast cancer cells Regulation.