miR-3074-5p及其靶基因p27在子痫前期胎盘组织中的异常表达

Dysregulated expressions of miR-3074-5p and its target gene p27 in placenta of pre-eclampsia patients

目的观察miR-3074-5p及其靶基因p27在子痫前期胎盘组织中的表达情况,初步探讨miR-3074-5p/p27分子途径在子痫前期病理过程中的作用。方法收集2017年9月至2018年3月期间在天津医科大学第二医院产科行剖宫产手术的子痫前期患者(子痫前期组,16例)和相同孕周非子痫前期的剖宫产产妇(对照组,9例)的胎盘组织,通过实时定量PCR、免疫组织化学(immunohistochemistry,IHC)和Western blotting检测,比较两组胎盘组织中miR-3074-5p以及p27、细胞周期蛋白D1(cyclin D1,CCND1)和细胞周期蛋白依赖性激酶2(cyclin dependent kinase 2,CDK2)等蛋白的表达水平。应用双荧光素酶基因报告系统,验证miR-3074-5p对p27 mRNA的靶向抑制作用;通过RNA干扰技术,敲低人绒毛外滋养细胞系HTR-8/SVneo中p27蛋白的表达水平,观察p27表达下调对细胞生理活性的影响。结果与对照组相比,子痫前期组胎盘组织中miR-3074-5p(P=0.034)与CCND1蛋白的表达量都显著下调(P=0.031),而p27蛋白的表达量则显著上调(P=0.010);IHC结果显示,p27和CCND1蛋白主要表达于合体滋养层细胞中。双荧光素酶报告系统检测结果证实,miR-3074-5p能与p27 mRNA的3’UTR序列结合而抑制其表达;敲低HTR-8/SVneo细胞中p27的表达水平后,细胞的增殖(P=0.014)和侵袭(P=0.045)活性都显著增强。结论miR-3074-5p可能通过直接靶向抑制p27的表达而参与调控人绒毛外滋养层细胞的生理活性,进而影响胎盘发育及功能;胎盘组织中miR-3074-5p的异常低表达可能通过诱导p27的表达而参与子痫前期的病理过程。

Objective To detect the expression levels of miR-3074-5p and its target gene p27 in the placental tissues of pre-eclampsia(PE)patients,and to preliminarily explore the association of miR-3074-5p/p27 pathway in the pathogenesis of PE.Methods From September 2017 to March 2018,16 pregnant women with PE(PE group)and 9 normal pregnant women(control group)were enrolled in Obstetrics Department in the Second Hospital of Tianjin Medical University.The placental expression levels of miR-3074-5p,p27,cyclin D1(CCND1)and cyclin dependent kinase 2(CDK2)were determined by quantitative PCR(qPCR),Western blotting and immunohistochemistry(IHC)analyses.The dual-luciferase reporter assay was applied to authenticate the directly targeted effect of miR-3074-5p on expression of p27 mRNA.The expression of p27 in the human extravillous trophoblast cell line HTR-8/SVneo was down-regulated by its specific shRNA,and the effects of down-regulated p27 expression on physiological activities of HTR-8/SVneo cells were detected.Results Compared with control group,the expression levels of miR-3074-5p(P=0.034)and CCND1(P=0.031)protein in the placenta of PE patients were significantly decreased,whereas the placental p27(P=0.010)protein expression was significantly increased in PE group.IHC analysis showed that,the signals of p27 and CCND1 proteins were dominantly detected in syncytiotrophoblast.The results of dual-luciferase reporter assay confirmed that miR-3074-5p could inhibit the expression of p27 by directly bound to the 3'UTR of p27 mRNA.After the p27 expression was down-regulated,the proliferative and invasive activities of HTR-8/SVneo cells were significantly enhanced(P=0.014,P=0.045).Conclusion miR-3074-5p might participate in the regulation of physiological activities of extravillous trophoblast cells by its targeted inhibition on p27 expression,and the abnormally decreased miR-3074-5p in trophoblast cells might be associated with the pathogenesis of PE by promoting the expression of p27.