摘要
目的探究微小RNA-138-5p(miR-138-5p)对滋养层细胞侵袭和血管生成的影响,并进一步研究其对缺氧诱导因子1α(HIF-1α)/Notch1轴的调节作用。方法体外培养人滋养层细胞系HTR-8/SVneo,分别转染mimics NC(miR-NC)、miR-138-5p模拟物(miR-138-5p mimics)、抑制剂阴性对照(inhibitor NC)、miR-138-5p抑制剂(miR-138-5p inhibitor)、pcDNA-NC(oe-NC)、pcDNA-HIF-1α(oe-HIF-1α)、miR-138-5p模拟物与pcDNA-NC(miR-138-5p mimics+oe-NC)、miR-138-5p模拟物与pcDNA-HIF-1α(miR-138-5p mimics+oe-HIF-1α)。转染后继续培养48 h,采用MTT法和克隆形成实验检测细胞增殖情况;Transwell法检测细胞侵袭情况;流式细胞术检测细胞凋亡率;管形成实验检测血管生成情况;qRT-PCR法检测细胞中miR-138-5p表达;Western blot法检测细胞中HIF-1α、Notch1、MMP-2、MMP-9、VEGF蛋白表达。双荧光素酶报告基因实验验证miR-138-5p与HIF-1α的靶向关系。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析和SNK-q检验。结果过表达miR-138-5p后细胞存活率、克隆形成数、侵袭个数、管形成数量(33.15±3.85比64.53±4.12)下降,细胞凋亡率升高,HIF-1α、Notch1、MMP-2、MMP-9、VEGF蛋白表达(0.26±0.04比0.46±0.05)降低(P均<0.05);抑制miR-138-5p表达上述指标变化相反。双荧光素酶报告基因实验结果显示miR-138-5p与HIF-1α存在靶向关系。过表达HIF-1α后细胞存活率、克隆形成数、侵袭个数、管形成数量升高,细胞凋亡率降低,Notch1及MMP-2、MMP-9、VEGF蛋白表达升高(P均<0.05);过表达HIF-1α可逆转过表达miR-138-5p对上述指标变化的影响。结论miR-138-5p可能通过靶向负调控HIF-1α/Notch1轴对滋养层细胞增殖、侵袭能力与血管生成产生影响。
Objective To explore the effects of microRNA-138-5p(miR-138-5p)on trophoblast invasion and angiogenesis,and to further study its regulatory impact on hypoxia-inducible factor 1α(HIF-1α)/Notch1 axis.Methods Human trophoblast cell line HTR-8/SVneo cultured in vitro were transfected with mimics NC(miR-NC),miR-138-5p mimics,inhibitor negative control(inhibitor NC),miR-138-5p inhibitor(miR-138-5p inhibitor),pcDNA-NC(oe-NC),pcDNA-HIF-1α(oe-HIF-1α),miR-138-5p mimics and pcDNA-NC(miR-138-5p mimics+oe-NC),miR-138-5p mimics and pcDNA-HIF-1α(miR-138-5p mimics+oe-HIF-1α),respectively.After transfection,the cells were cultured for 48 h,and the proliferation was detected by MTT assay and clone formation assay;cell invasion was detected by Transwell;the apoptosis rate was detected by flow cytometry;angiogenesis was detected by tube formation test;qRT-PCR detected the expression of miR-138-5p;Western Blot detected the expressions of HIF-1α,Notch1,MMP-2,MMP-9 and VEGF;dual luciferase reporter assay verified the targeting relationship between miR-138-5p and HIF-1α.Results The cell survival rate,the number of clone formations,the number of invasion and the number of tubes formed were significantly decreased after overexpression of miR-138-5p;In contrast,the apoptosis rate was significantly increased,and the protein expressions of HIF-1α,Notch1,MMP-2,MMP-9 and VEGF were significantly decreased(all P<0.05);inhibition of miR-138-5p expression changed the above indicators in the opposite direction.The results of the dual-luciferase reporter gene assay showed that miR-138-5p had a targeting relationship with HIF-1α.After HIF-1αoverexpression,the cell survival rate,the number of clone formation,the number of invasions and the number of tubes formed were significantly increased,the cell apoptosis rate was significantly decreased,and the expressions of Notch1,MMP-2,MMP-9 and VEGF were significantly increased(all P<0.05).Up-regulation of HIF-1αreversed the influence of miR-138-5p overexpression on the changes of the above indexe
作者
刘佳
付丽
杨月美
Jia Liu;Li Fu;Yuemei Yang(Department of Obstetrics,Caidian District People's Hospital,Wuhan,Hubei,430100)
出处
《中华细胞与干细胞杂志（电子版）》
2023年第5期277-287,共11页
Chinese Journal of Cell and Stem Cell（Electronic Edition）
