微小RNA-296对三阴性乳腺癌增殖与凋亡的影响

The effect of microRNA-296 on proliferation and apoptosis of triple negative breast cancer

目的检测微小RNA(miR)-296在三阴性乳腺癌(TNBC)组织及细胞株中的表达,探讨miR-296对TNBC细胞增殖,凋亡的影响及其机制。方法TNBC细胞株人乳腺癌细胞-231(MDA-MB-231),人乳腺癌细胞-453(MDA-MB-453)及人正常乳腺上皮细胞(MCF10A)购自中国科学院上海细胞生物学研究所,miR-296的过表达及干扰质粒分别转染至细胞株中,细胞分组为MDA-MB-231、MDA-MB-231-mimics、MDA-MB-231-anti-sh296及MDA-MB-453、MDA-MB-453-mimics、MDA-MB-453-anti-sh296;实时定量聚合酶链反应(Real-time PCR)检测miR-296在各组细胞中的相对表达量;使用LipofectamineTM 2000脂质体转染miR-296至细胞中,Real-time PCR证实转染成功后;通过细胞增殖-毒性检测试剂盒(CCK-8)检测对细胞生存率的影响;通过集落形成实验检测细胞的增殖能力;流式细胞实验检测miR-296对细胞凋亡的影响。应用SPSS 16.0统计软件分析。结果与癌旁组织比较,miR-296在TNBC组织中的含量明显降低(0.48±0.23比1.00±0.25,t=10.89,P<0.01)。miR-296在TNBC细胞株MDA-MB-231,MDA-MB-453中的表达明显低于正常乳腺上皮细胞MCF10A(0.19±0.08比1.00±0.26,t=8.36,P<0.01;0.46±0.19比1.00±0.26,t=4.68,P<0.01);与对照组比较,过表达miR-296后MDA-MB-231-mimics和MDA-MB-453-mimics细胞生存率低于转染前[(65.81±10.49)%比(99.39±22.58)%,t=3.82,P<0.01],[(73.17±11.81)%比(100.13±19.35)%,t=3.36,P<0.01];而给予干扰miR-296后细胞生存率较转染前升高;过表达miR-296后,细胞MDA-MB-231-mimics和MDA-MB-453-mimics的克隆相对值明显低于对照组(0.23±0.10比0.99±0.23,t=8.12,P<0.01),(0.43±0.16比1.01±0.20,t=6.44,P<0.01);而干扰miR-296后的克隆相对值明显高于对照组;过表达miR-296后,MDA-MB-231-mimics和MDA-MB-453-mimics细胞的凋亡百分数高于对照组[(25.76±2.83)%比(6.73±1.09)%,t=17.72,P<0.01],[(21.16±2.92)%比(5.56±0.87)%,t=14.47,P<0.01],干扰miR-296后细胞的凋亡百分数下降。过表达miR-296后,细胞MDA-MB-231-mimics和MDA-MB-453-的CX3趋化因子受体1(CX3CR1)的表达量低于对照组(0.36±0.14比1.00±0.20,t=7.45,P<0.01),0.43±0.10比1.00±0.16,t=8.48,P<0.01);而干扰miR-296后,细胞MDA-MB-231-sh-296和MDA-MB-453-sh-296的CX3CR1的表达量高于对照组(5.91±1.57比1.00±0.20,t=8.79,P<0.01),(4.89±1.08比1.00±0.16,t=10.03,P<0.01)。结论抑制miR-296的表达,可以通过靶向作用提高TNBC细胞的生存率,降低凋亡百分数,提示miR-296可能通过CX3CR1在TNBC中发挥抑癌作用。

Objective To investigate the effect and mechanism of microRNA(miR)-296 on proliferation and apoptosis of triple negative breast cancer(TNBC).Methods TNBC cell lines MDA-MB-231(human breast cancer cells-231),MDA-MB-453(human breast cancer cells-453)and MCF10A(human normal breast epithelial cells,Human normal mammary epithelial cells)was purchased from Shanghai Institute of Cell Biology,Chinese Academy of Sciences,and the overexpression and interference plasmids of miR-296 were transfected into cell lines respectively.The cells were divided into MDA-MB-231,MDA-MB-231-mimics,MDA-MB-231-anti-sh296,MDA-MB-453 and MDA-MB-453-mimics,MDA-MB-453-anti-sh296.Real-time PCR was used to detect the expression of miR-296 in MDA-MB-231,MDA-MB-453 and MCF10A cells.Then miR-296 mimics and inhibitors were transfected into MDA-MB-231 and MDA-MB-453 cells to up-regulate and inhibit the expression of miR-296;CCK-8 assay was applied to detect cell viability;colony formation assay was used to detect cell proliferation,after miR-296 was overexpressed;the apoptosis was detected by flow cytometry assay.Results Compared with para-cancer tissues,miR-296 in TNBC tissues was significantly decreased by 0.48±0.23 vs.1.00±0.25,t=10.89,P<0.01.The expression of miR-296 in TNBC cell lines MDA-MB-231 and MDA-MB-453 were significantly lower than those in normal mammary epithelial cells MCF10A(0.19±0.08 vs.1.00±0.26,t=8.36,P<0.01;0.46±0.19 vs.1.00±0.26,t=4.68,P<0.01).Compared with the control group,MDA-MB-231-mimics[(65.81±10.49)%vs.(99.39±22.58)%,t=3.82,P<0.01]and MDA-MB-453-mimics[(73.17±11.81)%vs.(100.13±19.35)%,t=3.36,P<0.01]cells survival rates were significantly lower than those before transfection.After interference of miR-296,MDA-MB-231 and MDA-MB-453 cells survival rates were higher than those before transfection.When miR-296 was overexpressed,MDA-MB-231(0.23±0.10 vs.0.99±0.23,t=8.12,P<0.01)and MDA-MB-453(0.43±0.16 vs.1.01±0.20,t=6.44,P<0.01)cells were significantly lower than those of the control group.However,interference of miR-296 MDA-MB-231 and MDA-MB-453 cells were significantly higher than those of the control group.After overexpression of miR-296,MDA-MB-231-mimics and MDA-MB-453-mimicscells apoptosis percentage were significantly increased[(25.76±2.83)%vs.(6.73±1.09)%,t=17.72,P<0.01],[(21.16±2.92)%vs.(5.56±0.87)%,t=14.47,P<0.01];after interference of miR-296,MDA-MB-231 and MDA-MB-453 cells percentage of apoptosis were decreased.After miR-296 was overexpressed,MDA-MB-231(0.36±0.14 vs.1.00±0.20,t=7.45,P<0.01)and MDA-MB-453(0.43±0.10 vs.1.00±0.16,t=8.48,P<0.01)cells expression level of CX3 chemokine receptor 1(CX3CR1)were significantly decreased;after interference of miR-296,MDA-MB-231(5.91±1.57 vs.1.00±0.20,t=8.79,P<0.01)MDA-MB-453(4.89±1.08 vs.1.00±0.16,t=10.03,P<0.01);cells expression level of CX3CR1 were increased.Conclusion Inhibiting the expression of miR-296 can improve the survival rate of TNBC cells and reduce the percentage of apoptosis,suggesting that miR-296 may play an anti-cancer role in TNBC through CX3CR1.