miR-21靶向E2F1对三阴性乳腺癌细胞恶性生物学活性及裸鼠肿瘤抑制率的影响

Effects of miR-21 targeting E2F1 on malignant biological activity of triple-negative breast cancer cells and its tumor inhibition rate in nude mice

目的探究miR-21靶向E2F1对三阴性乳腺癌细胞恶性生物学活性及裸鼠肿瘤抑制率的影响。方法将MDA-MB-231细胞分为5组,即MDA-MB-231组、miR-21 inhibitor组、miR-NC inhibitor组、siRNA-E2F1组和siRNA-NC组。检测细胞中miR-21表达(RT-PCR法);分别检测细胞增殖(MTT法)、侵袭(Transwell法)、迁移(划痕实验)和凋亡能力(流式细胞仪);检测细胞中E2F1蛋白表达;检测miR-21与E2F1的靶向关系(双荧光素酶实验报告)。结果MDA-MB-231细胞中miR-21表达明显高于MCF10A细胞(P<0.05);miR-21 inhibitor组细胞细胞中miR-21表达明显低于MDA-MB-231组(P<0.05)。与MDA-MB-231组相比,miR-21 inhibitor组细胞吸光度值、侵袭能力、迁移能力和细胞中E2F1蛋白表达均明显降低,细胞凋亡能力明显升高(P<0.05);MDA-MB-231组细胞吸光度值、侵袭、迁移、凋亡能力和细胞中E2F1蛋白表达与miR-NC inhibitor组相比差异无统计学意义(P>0.05)。预测软件显示E2F1的3′UTR端与miR-21有碱基互补结合点位。通过向MDA-MB-231细胞中转染野生型E2F1(E2F1-WT)时,miR-21组荧光素酶活性明显低于miR-NC组(P<0.05);miR-21组和miR-NC组突变体荧光素酶活性相比差异无统计学意义(P>0.05)。与siRNA-NC组相比,siRNA-E2F1组细胞增殖、侵袭、迁移和细胞中E2F1蛋白表达均明显降低,细胞凋亡能力明显增加(P<0.05)。与miR-NC inhibitor组裸鼠移植肿瘤第8天时相比,miR-21 inhibitor组裸鼠肿瘤体积明显降低,肿瘤抑制率为45.3%(P<0.05)。结论低表达miR-21可抑制三阴性乳腺癌细胞增殖、侵袭,促进凋亡,且抑制裸鼠移植瘤体积,其作用机制可能与抑制E2F1表达有关。

Objective To investigatethe effect of miR-21 targeting E2F1 on the malignant biological activity of triple-negative breast cancer cells and its tumor inhibition rate in nude mice.Methods The MDA-MB-231 cells were divided into 5 groups,including MDA-MB-231 group,miR-21 inhibitor group,miR-NC inhibitor group,siRNA-E2F1 group and siRNA-NC group.Th miR-21 expression in cells was assessed with RT-PCR method.The cell proliferation,invasion,migration and apoptosiswere assessed with MTT method,transwell method,scratch test and flow cytometry,respectively.The protein expression of E2F1 in cells was assessed.The targeting correlation between miR-21 and E2F1 was assessed dual-luciferase experimental report.Results The expression of miR-21 in MDA-MB-231 cells was significantly higher than that in MCF10A cells(P<0.05).The expression of miR-21 in miR-21 inhibitor group cells was significantly lower than that in MDA-MB-231 group(P<0.05).Compared with the MDA-MB-231 group,the absorbance,invasion ability,migration ability and E2F1 protein expression in the cells of the miR-21 inhibitor group were significantly reduced,while the apoptosis ability was significantly increased(P<0.05).Compared with the miR-NC inhibitor group,there was no significant differences in the absorbance,invasion,migration,apoptosis ability or E2F1 protein expression in the MDA-MB-231 group(P>0.05).The prediction software showed that the 3′UTR end of E2F1 had a base complementary binding site to miR-21.By transfecting wild-type E2F1(E2F1-WT)into MDA-MB-231 cells,the luciferase activity of miR-21 group was significantly lower than that of miR-NC group(P<0.05).There was no significant difference in luciferase activity between miR-21 group and miR-NC group(P>0.05).Compared with the siRNA-NC group,the cell proliferation,invasion,migration and E2F1 protein expression in the siRNA-E2F1 group were significantly reduced,and the apoptosis ability was significantly increased(P<0.05).Compared with the miR-NC inhibitor group on the 8th day of tumor transplantation,the tumor volume of the miR-21 inhibitor group was significantly reduced,and the tumor inhibition rate was 45.3%(P<0.05).Conclusion Low expression of miR-21 can inhibit the proliferation and invasion of triple-negative breast cancer cells,promote apoptosis,and inhibit the volume of transplanted tumors in nude mice,and its mechanism may be related to the inhibition of E2F1 expression.