摘要
探究miR-383靶向调控人可溶性血管内皮生长因子受体-1(sFlt-1)在子痫前期滋养细胞发生发展中的分子生物学机制。将含有野生型和突变型miR-383结合位点的sFlt-1序列克隆到萤光素酶报告载体中,并在HEK-293T细胞中与miR-383 mimics共转染,用双萤光素酶进行基因检测。用miR-383 mimic、miR-383 inhibitors及其阴性对照质粒对HTR-8/SVneo细胞进行转染,通过qRT-PCR和Western Blot研究miR-383表达及对sFlt-1表达水平的影响,CCK-8法用于检测滋养细胞的增殖,Transwell法用于分析滋养细胞的侵袭和迁移能力。双荧光素酶检测结果显示:在人滋养细胞中,miR-383通过结合sFlt-1基因靶序列而抑制sFlt-1基因表达。在抑制miR-383表达后,可增加sFlt-1 mRNA的转录水平和蛋白表达水平,但降低了人滋养细胞的增殖、迁移以及侵袭水平;而在增加miR-383表达后,sFlt-1 mRNA的转录水平和蛋白表达水平降低,人滋养细胞的增殖、迁移以及侵袭水平明显提高。在子痫前期滋养细胞中,miR-383可靶向结合sFlt-1基因而抑制其转录表达,下调翻译水平,进而降低滋养细胞增殖、迁移和侵袭能力。
miR-383 is aberrantly expressed in patients with severe preeclampsia and can serve as a biomarker for the diagnosis and disease assessment of metastasis in severe preeclampsia.This study aims to investigate the molecular mechanism by which miR-383regulates the development of trophoblasts in preeclampsia through targeted modulation of human soluble vascular endothelial growth factor receptor-1(sFlt-1).To confirm the direct targeting of sFlt-1 by miR-383,DNA sequences containing either the wild-type or mutant miR-383 binding site from the sFlt-1 gene were cloned into a luciferase reporter vector.The constructs were co-transfected with miR-383 mimics into HEK-293T cells,and the interaction was assessed using a dual-luciferase assay.To investigate the functional role of miR-383,HTR-8/SVneo trophoblast cells were transfected with miR-383 mimic,miR-383 inhibitor,or their respective negative controls.The effects on miR-383 and sFlt-1 expression levels were evaluated by qRT-PCR and Western blot.Furthermore,trophoblast cell proliferation,migration,and invasion were analyzed using the CCK-8 and Transwell assays,respectively.The dualluciferase assay in HEK-293T cells demonstrated that miR-383 significantly reduced the luciferase activity of the wild-type sFlt-1reporter construct by binding to its specific target sequence,whereas it had no significant effect on the mutant construct.This confirms the direct targeting of sFlt-1 by miR-383.Subsequently,functional experiments in HTR-8/SVneo trophoblasts revealed that miR-383inhibition upregulated both sFlt-1 mRNA and protein levels but suppressed cellular proliferation,migration,and invasion.Conversely,miR-383 overexpression downregulated sFlt-1 and markedly enhanced these critical cellular functions.In preeclamptic trophoblasts,miR-383 can target the sFlt-1 gene,thereby inhibiting its transcription and reducing its translation levels,which subsequently diminishes the cells'abilities to proliferate,migrate,and invade.After an increase in miR-383 expression,a further reduction was observed in both the transcriptional and translational levels of the sFlt-1 gene,as well as in the levels of proliferation,migration,and invasion.
作者
沈树娜
沈婕
李炼
邓森灵
林元
SHEN Shuna;SHEN Jie;LI Lian;DENG Senling;LIN Yuan(Department of Obstetrics and Gynecology,Western Hainan Central Hospital,Danzhou 571700,China)
出处
《药物生物技术》
2025年第6期753-759,共7页
Pharmaceutical Biotechnology
基金
海南省卫生健康科技创新联合项目(No.WSJK2024MS221)。
