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

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.