Effects of Gadd45α on cell migration and invasion in the human trophoblast derived HTR8/SVneo cells

Liu Xiru; Mu Huaqiao; Luo Xin; Qi Hongbo

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Effects of Gadd45α on cell migration and invasion in the human trophoblast derived HTR8/SVneo cells

更新时间：2023-12-18

- Effects of Gadd45α on cell migration and invasion in the human trophoblast derived HTR8/SVneo cells
- Journal of Chongqing Medical University Vol. 38, Issue 11, Pages: 1516-1521(2014)
- 作者机构：
  
  1. 重庆医科大学附属第一医院妇产科,重庆,400016
  2. 重庆市妇幼保健院医务科,重庆,400013
- 作者简介：
- 基金信息：
- DOI：
  CLC：
- Published：2014，
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Liu Xiru, Mu Huaqiao, Luo Xin, et al. Effects of Gadd45α on cell migration and invasion in the human trophoblast derived HTR8/SVneo cells. [J]. Journal of Chongqing Medical University 38(11):1516-1521(2014)
DOI：

Liu Xiru, Mu Huaqiao, Luo Xin, et al. Effects of Gadd45α on cell migration and invasion in the human trophoblast derived HTR8/SVneo cells. [J]. Journal of Chongqing Medical University 38(11):1516-1521(2014) DOI：

摘要

目的：研究人生长阻滞和DNA损伤45α（growth arrest and DNA damage 45 alpha，Gadd45α）基因对滋养细胞HTR8/SV－neo增殖、凋亡、迁移和侵袭等生物学功能的影响，探讨其在子痫前期（preeclampsia，PE）发生发展中的可能作用。方法：构建Gadd45α短发夹干扰RNA，以阴性对照组作为参照，转染人滋养细胞HTR8/SVneo，即分为实验组（si-Gadd45α）和阴性对照组（si-NC）进行实验；应用流式细胞仪检测转染效率，并应用qRT-PCR和Western blot检测转染后各组细胞中Gadd45α mRNA和蛋白表达水平；采用四甲基偶氮唑蓝（methyl thiazolyl tetrazolium，MTT）显色法检测转染后各组细胞增殖能力；应用流式细胞仪对转染后各组细胞进行细胞凋亡分析；采用Transwell法检测转染后各组细胞迁移和侵袭能力；采用早孕绒毛外植体培养模型观察敲除Gadd45α基因后对绒毛外滋养细胞外生性迁移能力的影响；收集各组细胞培养上清液，明胶酶谱法检测基质金属蛋白酶（matrix metalloproteinases，MMPs）的表达，蛋白免疫印迹检测基质金属蛋白酶组织抑制因子（tissue inhibitors of MMPs，TIMPs）的表达。结果：流式细胞仪检测转染效率约为 90％；转染后实验组较对照组Gadd45α mRNA的表达量约降低80%，蛋白表达水平约下降70%，差异均有统计学意义（P<0.01），证明干扰Gadd45α表达成功。MTT法和流式细胞仪检测细胞增殖和凋亡，结果显示实验组和对照组细胞增殖和凋亡均无统计学差异（P >0.05）。Transwell实验表明干扰Gadd45α后HTR8/SVneo侵袭能力明显增加，约为对照组的1.65倍；迁移能力也明显增加，约为对照组的2倍，均有统计学差异（P<0.01）。早孕绒毛外植体培养结果显示：与阴性对照组的绒毛相比，Gadd45α干扰片段处理的绒毛外滋养细胞外生性迁移的距离明显增加，差异有统计学意义（P=0.005）。明胶酶谱实验结果显示干扰Gadd45α后基质金属蛋白酶-2（matrix metalloproteinases，MMP-2）和MMP-9的活性增强，而Western blot检测发现其抑制因子TIMP-1和TIMP-2相应地下降（P<0.01）。结论：推测Gadd45α可能是通过调控蛋白酶的活性来抑制滋养细胞的迁移和侵袭，从而参与PE的发生发展。

Abstract

Objective：To investigate the effects of growth arrest and DNA damage 45 alpha（Gadd45α） gene on pro1iferation，apopto－sis，migration and invasion of HTR8/SVneo cells and the role of Gadd45α in the pathogenesis of preeclampsia. Methods：The role of Gadd45α in HTR8/SVneo cells was examined by RNA interference technology using Gadd45α specific short hairpin RNA（siRNA）. The experiment was composed of two groups：si-Gadd45α group and si-negative control group（si-NC group）. mRNA and protein ex－pression of Gadd45α was identified by qRT-PCR and Western blot respectively. Changes of cell proliferation，apoptosis，migration and invasion were respectively detected by methyl thiazolyl tetrazolium（MTT），flow cytometry，transwell migration and Matrigel invasion assay. Placental villious explants culture model was employed to further verify the effect of Gadd45α on outgrowth and migration of extravillous trophoblast cells. Gelatin zymography was used to detect the expression of matrix metalloproteinase（MMP）-2/9 in culture medium. Western blot was used to detect the expressions of tissue inhibitors of MMPs（TIMP）-1 and TIMP-2. Results：（1）The trans－fection efficiency was about 90%. The mRNA and protein levels of Gadd45α in si-Gadd45α group were reduced by 80% and 70% respectively，with significant differences（P<0.01）. （2）The results of MTT and flow cytometry indicated that cell prolifer－ation and apoptosis were not affected by siRNA of Gadd45α（P＞0.05）. （3）Knocking-down Gadd45α expression by siRNA significantly promoted invasion（1.65 folds） and migration（2 folds） potentials of HTR8/SVneo cells（P<0.01）. （4）Gadd45α silencing significantly promoted outgrowth of villous explants in vitro（P=0.005）. （5）Gadd45α regulated migration and invasion through control－ling gelatinolytic activities of MMP-9，MMP-2（P<0.05） and the expressions of TIMP-1 and TIMP-2（P<0.01）. Conclusion：Gadd45α is probably involved in the onset of preeclampsia by regulating invasion and migration of trophoblast through controlling the activities of MMPs and the expressions of TIMPs.

关键词

生长阻滞和DNA损伤45α基因滋养细胞侵袭滋养细胞迁移子痫前期

Keywords

growth arrest and DNA damage 45 alphatrophoblast invasiontrophoblast migrationpreeclampsia

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Laboratory of Reproductive Biology,School of Public Health and Management,Chongqing Medical University,Canada-China-New Zealand Joint Laboratory of Maternal and Fetal Medicine of Chongqing Medical University

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