慢病毒介导的RNA干扰沉默RRS1基因对乳腺癌细胞MCF-7生物学行为的影响

EFFECT OF LENTIVIRUS-MEDIATED RNA INTERFERENCE SILENCING OF RRS1 GENE ON BIOLOGICAL BEHAVIOR OF BREAST CANCER MCF-7 CELLS

目的探讨慢病毒介导的RNA干扰技术抑制RRS1表达对人乳腺癌细胞MCF-7生物学行为的影响。方法采用Western blot方法检测RRS1在MCF-7与正常乳腺上皮细胞HMEC中的表达;分别构建携带绿色荧光蛋白的shRNA-RRS1慢病毒和阴性对照慢病毒RNA干扰系统,分别感染MCF-7细胞,即得到shRRS1实验组和shCtrl阴性对照组MCF-7细胞;采用荧光定量PCR和Western blot方法分别检测两组细胞的mRNA及蛋白表达,确定敲减效率;采用MTT法连续6d检测两组细胞增殖活力;采用PI染色流式细胞术检测两组细胞的细胞周期分布;采用DAPI荧光染色、Annexin V-APC单染流式细胞术及Caspase 3/7实验检测两组细胞凋亡情况;采用Transwell实验检测两组细胞迁移能力的变化。结果实验组中RRS1的表达明显高于HMEC(P<0.05);与对照组相比,实验组RRS1mRNA和蛋白水平均明显降低(P<0.05),细胞活力显著减弱(P<0.05),G1期细胞明显增加(P<0.05),且实验组细胞发生典型的凋亡形态学改变,凋亡率显著增高(P<0.05),细胞迁移能力显著降低(P<0.05)。结论 RRS1在乳腺癌细胞MCF-7中高表达。敲减RRS1基因能明显诱导乳腺癌细胞凋亡及周期阻滞,同时使得细胞迁移能力明显降低。

Objective To investigate the effect of RRSl inhibition by lentivirus-mediated RNA interference technique on the biological behavior of human breast cancer MCF-7 cells.Methods Western blot was used to measure the expression of RRS1 in MCF-7 cells and normal human mammary epithelial cells(HMECs).The shRNA-RRSl lentivirus carrying green fluorescent protein and alentivirus RNA interference system for negative control were established and used to infect MCF-7 cells to obtain the MCF-7 cells in shRRSl experimental group and shCtrl negative control group.Real time PCR and Western blot were used to measure the mRNA and protein expression of RRSl and determine the efficiency of knock-down.MTT assay was used to measure cell proliferation in 6 consecutive days:PI flow cytometry was used to analyze the distribution of cell cycles in the twogroups:DAP I fluorescent staining,Annexin V-APC singledye FCM.and caspase-3/7 assay were used to measure cell apoptosis;Transwell assay was used to observe the change in cell migration ability.Results MCF-7 cells had significantly higher expression of RRS1 than HMECs(P<0.05).Compared with the shCtrl control group,the shRRSl experimental group had significant reductions in the mRNA and protein expression of RRS1(P<0.05),a significant reduction in cell viability(P<0.05),and a significant increase in the proportion of cells arrested in G1 phase(P<0.05),as well as typical morphological changes of apoptosis,a significant increase in apoptosis rate,and a significant reduction in cell migration ability(P<0.05).Conclusion RRS1 is highly expressed in breast cancer MCF-7cells.Knock-down of the RRSl gene can induce apoptosis and cycle of breast caneer cells lead to a significant reduction in cell migration ability.