慢病毒介导的ROBO1沉默抑制乳腺癌细胞生长克隆及ATP合成

摘要

目的 研究沉默ROBO1对乳腺癌细胞生长、克隆及三磷酸腺苷(ATP)合成的影响.方法 乳腺癌细胞MDA-MB-231感染ROBO1 siRNA重组病毒和siRNA control重组病毒记为干扰组和阴性组,以不做处理的细胞作为对照组.qRT-PCR和Western blot检测细胞中ROBO1 mRNA和蛋白水平,以MTT法检测细胞增殖情况,克隆形成实验检测细胞克隆形成能力,荧光素酶法检测细胞合成的ATP水平,流式细胞术检测细胞周期,Western blot检测细胞中周期蛋白D1(CyclinD1)、p21蛋白水平.结果 阴性组细胞中ROBO1 mRNA和蛋白水平、细胞OD值、克隆形成率、ATP水平、细胞周期比率及cyclinD1、p21蛋白水平与对照组比较,差异均无统计学意义(P ＞0.05).干扰组细胞中ROBO1 mRNA和蛋白水平降低,细胞OD值也降低,克隆形成率及ATP水平下降,G0/G1期细胞比率升高,细胞中cyclinD1蛋白水平降低,p21蛋白水平升高,与对照组比较,差异有统计学意义(P ＜0.05).结论 沉默ROBO1抑制乳腺癌细胞的生长、克隆及ATP合成能力,并且能将细胞周期阻滞在G0/G1期.%Objective To study the effect of silencing ROBO1 on the growth,cloning and ATP synthesis of breast cancer cells. Methods Breast cancer cell MDA-MB-231 infected with ROBO1 siRNA recombinant virus and siRNA control recombinant virus were recorded as inter-ference group and negative group,the cells those were not treated were used as the control group. qRT-PCR and Western blot were used to detect the level of ROBO1 mRNA and protein in cells,cell proliferation was detected by MTT method,colony forming assay was used to detect cell clone formation ability,the ATP level of cell synthesis was detected by luciferase method,cell cycle was detected by flow cytometry,Western blot was used to detect the level of cyclinD1 and p21 protein in cells. Results The levels of ROBO1 mRNA and protein,cell OD value,clone formation rate,ATP level,cell cycle ratio and cyclinD1,p21 protein levels in the negative group were compared with those of the control group,the differ-ence was not statistically significant (P ＜0.05). The level of ROBO1 mRNA and protein in the interference group decreased,OD value de-creased,the rate of clone formation and the level of ATP decreased,the proportion of cells in G0/G1phase increased,the level of cyclinD1 protein in the cells decreased,the level of p21 protein was elevated,compared with the control group,the difference was statistically significant (P ＜0.05). Conclusion Silencing ROBO1 inhibits the growth,cloning and ATP synthesis ability of breast cancer cells,and the cell cycle was blocked in G0/G1phase.