Ni Huijie, Wang Xiao, Qu Hongmei, Gao Xueli, Yu Xiaoyan
Department of Obstetrics, the Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China.
J Obstet Gynaecol Res. 2021 Jan;47(1):184-197. doi: 10.1111/jog.14451. Epub 2020 Sep 13.
Low density lipoprotein receptor-related protein 6 (LRP6) has been demonstrated to control trophoblast cell invasion, but its regulatory gene remains undefined. In this study, microRNA (miR) regulating LRP6 were explored to elucidate the potential mechanism of preeclampsia (PE).
Firstly, the expression of LRP6 in PE tissues was detected by immunohistochemical staining and quantitative real-time polymerase chain reaction (qRT-PCR) assay. Prediction software predicted that LRP6 might be the target gene of miR-95-5p, and verified by double-luciferase reporter analysis. qRT-PCR assay measured the expression of miR-95-5p in PE tissues and trophoblast cell lines. Then, we transfected miR-95-5p mimic, inhibitor, LRP6, or mimic plus LRP6 into trophoblast cell lines, and analyzed their influences on cell migration and invasion by wound healing and Transwell experiments. The expressions of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors of metalloproteinase (TIMP)-1 in transfected cells were examined by western blot (WB) analysis.
LRP6 was low-expressed in PE tissues, while miR-95-5p expression was high-expressed. MiR-95-5p negatively regulated the LRP6 expression in trophoblast cells. Both up-regulated LRP6 and down-regulated miR-95-5p can not only promote the migration and invasion of trophoblast cells, but also raised the expressions of MMP-2 and MMP-9 and inhibited the expression of TIMP-1. The over-expression of miR-95-5p suppressed the metastasis of trophoblast cells and rescued LRP6-induced increase of MMP-2 and MMP-9 and reduction of TIMP-1.
MiR-95-5p involved in the migration and invasion of trophoblast cells by targeting LRP6, which might be a potential therapeutic target for PE.
低密度脂蛋白受体相关蛋白6（LRP6）已被证明可控制滋养层细胞侵袭，但其调控基因仍不明确。本研究旨在探索调控LRP6的微小RNA（miR），以阐明子痫前期（PE）的潜在机制。
首先，通过免疫组织化学染色和定量实时聚合酶链反应（qRT-PCR）检测PE组织中LRP6的表达。预测软件预测LRP6可能是miR-95-5p的靶基因，并通过双荧光素酶报告基因分析进行验证。qRT-PCR检测PE组织和滋养层细胞系中miR-95-5p的表达。然后，将miR-95-5p模拟物、抑制剂、LRP6或模拟物加LRP6转染到滋养层细胞系中，并通过伤口愈合和Transwell实验分析它们对细胞迁移和侵袭的影响。通过蛋白质免疫印迹（WB）分析检测转染细胞中基质金属蛋白酶（MMP）-2、MMP-9和金属蛋白酶组织抑制剂（TIMP）-1的表达。
LRP6在PE组织中低表达，而miR-95-5p表达高。miR-95-5p在滋养层细胞中负调控LRP6表达。上调LRP6和下调miR-95-5p不仅能促进滋养层细胞的迁移和侵袭，还能提高MMP-2和MMP-9的表达并抑制TIMP-1的表达。miR-95-5p的过表达抑制了滋养层细胞的转移，并挽救了LRP6诱导的MMP-2和MMP-9增加以及TIMP-1减少。
miR-95-5p通过靶向LRP6参与滋养层细胞的迁移和侵袭，这可能是PE的一个潜在治疗靶点。
