微RNA-378a-3p靶向SUFU基因促进乳腺癌细胞的增殖和迁移

Micro RNA-378a-3p targeting SUFU gene promotes proliferation and migration of breast cancer cells

目的:研究微RNA(microRNA,miRNA,miR)-378a-3p对乳腺癌细胞体外增殖、凋亡和迁移能力的影响,以及可能的作用机制。方法:利用GEO(Gene Expression Omnibus)数据库分析乳腺癌miR-378a-3p高表达患者与低表达患者的生存情况。使用实时荧光定量PCR法检测人乳腺癌细胞系以及正常人乳腺上皮细胞中miR-378a-3p的表达水平。采用慢病毒感染的方法将携带miR-378a-3p-模拟物(miR-378a-3p-mimics)的重组慢病毒转入人乳腺癌MCF-7细胞,构建稳定过表达miR-378a-3p的MCF-7细胞;将携带miR-378a-3p-抑制子(miR-378a-3p-inhibitor)的重组慢病毒转入人乳腺癌MDA-MB-231细胞,构建稳定抑制miR-378a-3p表达的MDA-MB-231细胞。采用CCK-8法、Transwell小室法和FCM法检测过表达miR-378a-3p或抑制其表达对乳腺癌细胞增殖、凋亡和迁移的影响。通过生物信息学分析以及双荧光素酶报告系统验证miR-378a-3p对其靶基因SUFU(Hedgehog信号通路负调控因子)的直接靶向调控作用。miR-378a-3p表达水平改变后,通过免疫共沉淀法验证SUFU蛋白与人GLI家族锌指蛋白1(GLI family zinc finger 1,GLI1)之间的相互作用,并采用蛋白质印迹法检测2者在细胞核和细胞质中表达水平的变化情况。在过表达miR-378a-3p或抑制其表达的基础上,使用Hedgehog信号通路抑制剂或激活剂处理乳腺癌细胞,然后采用CCK-8法、Transwell小室法和FCM法检测过表达miR-378a-3p或抑制其表达是否通过激活或抑制Hedgehog信号通路调控乳腺癌细胞的增殖、凋亡和迁移。结果:高表达miR-378a的乳腺癌患者的预后较低表达miR-378a的乳腺癌患者的差。miR-378a-3p在多种乳腺癌细胞中的表达水平均明显上调(P值均<0.001)。乳腺癌MCF-7细胞稳定过表达miR-378a-3p后,MCF-7细胞的体外增殖(P<0.05)和抗凋亡(P<0.01)能力均明显增强;反之,在乳腺癌MDA-MB-231细胞中沉默miR-378a-3p表达后,MDA-MB-231细胞的体外增殖(P<0.000 1)、抗凋亡(P<0.05)和迁移(P<0.001)能力均明显降低。乳腺癌细胞高表达miR-378a-3p后,能够通过抑制SUFU的表达,削弱SUFU与GLI1的相互作用,促使细胞核内GLI1含量增加,从而激活Hedgehog信号通路,提高乳腺癌细胞的增殖、抗凋亡和迁移能力。结论:miR-378a-3p过表达通过负调控SUFU以激活Hedgehog信号通路,增强人乳腺癌细胞的增殖、抗凋亡和迁移能力,发挥促癌作用。

Objective:To investigate the effects of microRNA (microRNA,miRNA,miR)-378a-3p on the proliferation,apoptosis and migration of breast cancer cells in vitro,and the possible mechanisms.Methods:The GEO (Gene Expression Omnibus) database was used to analyze the survival of breast cancer patients with high or low expression of miR-378a-3p.The real-time fluorescent quantitative PCR was performed to detect the expression level of miR-378a-3p in human breast cancer cell lines and normal human breast epithelial cells.A recombinant lentivirus carrying miR-378a-3p-mimics was transfected into human breast cancer MCF-7 cells using a lentiviral infection method to construct MCF-7 cells that stably overexpress miR-378a-3p,and a recombinant lentivirus carrying miR-378a-3p-inhibitor was transferred into human breast cancer MDA-MB-231 cells to construct MDA-MB-231 cells that stably inhibit miR-378a-3p expression.The effects of overexpression of miR-378a-3p or inhibition of its expression on proliferation,apoptosis and migration of breast cancer cells were examined by CCK-8 assay,Transwell assay and FCM.The direct targeting and regulation of miR-378a-3p on its target gene SUFU—a negative regulator of Hedgehog signaling pathway,was verified by bioinformatics analysis and dual luciferase reporter system.After the altered expression of miR-378a-3p,the interaction between SUFU protein and human GLI family zinc finger 1 (GLI1) protein was verified by immunoprecipitation,and the altered expression levels of both proteins in the nucleus and cytoplasm were detected by Western blotting.On the basis of overexpression of miR-378a-3p or inhibition of its expression,breast cancer cells were treated with Hedgehog signaling pathway inhibitor or activator,followed by CCK-8 assay,Transwell assay and FCM to detect whether the overexpression of miR-378a-3p or inhibition of its expression regulated the proliferation,apoptosis and migration of breast cancer cells through activation or inhibition of Hedgehog signaling pathway.Results:The prognosis of breast cancer patients with high expression level of miR-378a was worse than that of those with low expression level.The expression of miR-378a-3p was significantly upregulated in a variety of breast cancer cells (all P < 0.001).After stable overexpression of miR-378a-3p in MCF-7 cells,the proliferation (P < 0.05) and anti-apoptosis (P < 0.01) abilities of MCF-7 cells were significantly improved.Conversely,after silencing miR-378a-3p expression in MDA-MB-231 cells,the proliferation (P < 0.000 1),anti-apoptosis (P < 0.05) and migration (P < 0.001) abilities of MDA-MB-231 cells were significantly reduced.The high expression of miR-378a-3p in breast cancer cells promoted the proliferation,anti-apoptosis and migration abilities of breast cancer cells by inhibiting the expression of SUFU,weakening the interaction between SUFU and GLI1,and leading to an increase in GLI1 content in the nucleus,thereby activating the Hedgehog signaling pathway.Conclusion:Overexpression of miR-378a-3p negatively regulates the expression of SUFU to activate the Hedgehog signaling pathway and enhance the proliferation,anti-apoptosis and migration capabilities of human breast cancer cells to promote tumor progression.