miR-30c-5p通过调节Notch1的表达抑制人绒毛膜滋养层细胞HTR-8/SVneo的迁移、侵袭和管状形成

Inhibition of the migration,invasion,and tube formation of human chorionic trophoblast cells HTR-8/SVneo by miR-30c-5p regulating Notch1 expression

目的:探讨miR-30c-5p通过调节Notch1表达对人绒毛膜滋养层细胞HTR-8/SVneo迁移、侵袭和管状形成调控的机制,确定其在子痫前期发病中的潜在作用。方法:在体外对人绒毛膜滋养层细胞HTR-8/SVneo进行培养,慢病毒系统构建miR-30c-5p过表达及敲低稳转HTR-8/SVneo细胞系。分为对照组、导入miR-30c-5p过表达质粒组和导入miR-30c-5p敲低质粒组。通过荧光定量PCR(qRT-PCR)检测各组细胞中miR-30c-5p和Notch1基因的表达量,蛋白质免疫印迹法(Western blot)检测各组细胞中Notch1蛋白的表达量,划痕愈合实验、Transwell侵袭实验和管状形成实验分别检测各组细胞的迁移、侵袭和管状形成能力。结果:与对照组比较,miR-30c-5p过表达质粒组中细胞的miR-30c-5p基因表达水平升高(P<0.0001),细胞Notch1蛋白表达水平下降(P<0.01),细胞迁移(P<0.01)、侵袭(P<0.001)和管状形成能力(P<0.05)均受到抑制。反之,miR-30c-5p敲低质粒组中细胞的miR-30c-5p基因表达水平下降(P<0.01),细胞Notch1蛋白表达升高(P<0.001),细胞的迁移(P<0.01)、侵袭(P<0.05)以及细胞的管状形成能力(P<0.01)增强。结论:miR-30c-5p能够通过抑制Notch1的表达降低人绒毛膜滋养层细胞HTR-8/SVneo的迁移、侵袭和管状形成能力,参与子痫前期的发病。

Objective:To explore the mechanism of the inhibition of the migration,invasion,and tube formation of hu-man chorionic trophoblast cells HTR-8/SVneo(HTR-8/SVneo)by miR 30c-5p regulating Notch1 expression,and to determine the potential role of miR-30c-5p in the pathogenesis of preeclampsia.Methods:The HTR-8/SVneo cells were cultured in vitro.The HTR-8/SVneo cell lines with miR-30c-5p overexpression or knockdown stabilized were constructed by lentivirus system.The HTR-8/SVneo cells lines were divided into group A(control group),group B(Ccells lines with miR 30c-5p overexpression),and group C(cells lines with miR-30c-5p knockdown).The gene expres-sions of miR-30c-5p and Notch1 of the cells in the three groups were detected by quantitative real-time PCR(qRT-PCR).The expressions of Notch1 protein of the cells in the three groups were detected by Western blot.The migration,invasion,and tube formation ability of the cells in the three groups were detected by wound healing assay,Tran-swell method,and tube formation assay,respectively.Results:The expression level of miR-30c-5p gene of the cells in group B was significantly higher than that of the cells in group A(P<0.0001),the expression level of Notch1 protein of the cells in group B was significantly lower than that of the cells in group A(P<0.01),and the abilities of migra-tion,invasion,and tubular formation of the cells in group B were significantly lower than those of the cells in group A(P<0.01,P<0.001,and P<0.05).The expression level of miR-30c-5p gene of the cells in group C was significantly lower than that of the cells in group A(P<0.0001),the expression level of Notch1 protein of the cells in group C was significantly higher than that of the cells in group A(P<0.001),and the abilities of migration,invasion,and tubular formation of the cells in group C were significantly higher than those of the cells in group A(P<0.01,P<0.05,and P<0.01).Conclusion:MiR-30c-5p can decrease migration,invasion,and tube formation of HTR-8/SVneo cells by in-hibiting the expression of Notch1,which is involved in the pathogenesis of preeclampsia.