Study Of Rac1 Signaling Pathway On The Role And Mechanism Of Trophoblast Invasion

Part ⅠEffect of Racl Signalling Pathway on Normal Pregnancy and PreeclampsiaObjectiveTo investigate the mRNA and protein expression of Racl Signalling Pathway in normal pregnancy and preeclampsia as well as its relation to the trophoblast invasiveness.MethodsA total of 117 subjects, who were in different trimesters of pregnancy, were recruited from outpatient and inpatient services at the Department of Obstetrics and Gynecology, the Second Hospital of Shandong University, Jinan, from June 2010 to December 2014. They were 25 first trimester subjects at 6-9 weeks of gestation (average age:28.5 ± 3.8 years),25 term gestation subjects at 37-40 weeks of gestation (average age:27.9 ± 3.3 years), and 67 patients with preeclampsia at 37-40 weeks of gestation. The preeclampsia cohort included 37 women with mild preeclampsia (average age:28.9 ± 3.25 years) and 30 with severe forms of preeclampsia (average age:26.9 ± 2.8 years). The trophoblast samples from the first trimester of normal pregnancies were collected during induced abortion procedures, while the term pregnancy and preeclampsia placental tissues from the third trimester were obtained by caesarean section. We further measured the activity of Racl and its downstream targets P-catenin, Snail and MMP9 in placental tissues from normal pregnancy and preeclampsia by Real-time PCR, immunohistochemistry, GTPase pull down and Western blot analysis.ResultsIn this study, we investigated Rac1 activation in early pregnancy, term pregnancy and preeclampsia placental tissues using GTPase pull down and Western blot. The findings demonstrated that GTP-Rac1 expression level in early pregnancy placental tissues was significantly higher than in term pregnancy (P<0.05). Lower levels were examined in mild preeclampsia and severe preeclampsia than in term pregnancy (P<0.05); and GTP-Racl expression level in severe preeclampsia significantly reduced than mild preeclampsia; in particular, no significant differences in Racl protein levels were observed in all placental tissues (P>0.05). Western blot analysis demonstrated that intranuclear β-catenin and Snail expression levels in normal early pregnancy placental tissues were significantly higher than term pregnancy (P<0.05). Expression Levels in mild preeclampsia and severe preeclampsia markedly reduced compared with normal term pregnancy and there were statistically significant differences (P<0.05). Compared to mild preeclampsia, expression levels were lower in severe preeclampsia (P<0.05). Immunohistochemistry showed that MMP9 in villous stroma were little expressed, suggesting that MMP9 was mainly distributed in cytoplasm and it was claybank or dark claybank. Compared to normal term pregnancy, the number of MMP9 positive cells was significantly increased in early pregnancy (P<0.05), whereas the number of cells with weaker staining was lower in mild preeclampsia and severe preeclampsia and there were statistical significant differences (P<0.05). MMP9 expression level in severe preeclampsia was markedly lower than mild preeclampsia (P<0.05). Western blot and Real-time PCR analysis revealed that expression levels of MMP9 protein and mRNA in early pregnancy placental tissues were higher than normal term pregnancy (P<0.05). Lower levels were found in mild preeclampsia and severe preeclampsia compared to normal term pregnancy placental tissues (P<0.05); in particular, levels in severe preeclampsia placental tissues were significantly decreased compared to mild preeclampsia (P<0.05).Conclusion(1) The activation of Racl in normal pregnancy placental tissues decreased with gestational weeks and GTP-Racl expression level in term pregnancy was significantly lower than early pregnancy. The expressions of Racl downstream target genes β-catenin and Snail as well as MMP9 in placental tissues closely related to trophoblasts invasiveness were consistent with the variation of Racl activation. Moreover, the variation of Racl signaling pathway was the same as the changes that trophoblasts invasiveness was the strongest in early pregnancy and the weakest in term pregnancy, suggesting that Racl signaling pathway may regulate MMP9 expression and trophoblast invasion in normal pregnancy.(2) Compared to normal term pregnancy, expression levels of GTP-Rac1, intranuclear β-catenin and Snail as well as MMP9 significantly reduced in preeclampsia placental tissue. Expression levels were the lowest in severe preeclampsia, suggesting that Racl signaling pathway may play a critical role in preeclampsia placental shallow invasion by regulating MMP9 expression.Part ⅡEffect of Rac1/β-catenin Pathway on the behavior of extravillous trophoblast invasionObjectiveTo discuss the roles of Racl signaling pathway in MMP9 activation and trophoblast invasion of human extravillous trophoblasts HTR-8/SVneo in vitroMethodsTo cultivate human extravillous trophoblasts HTR-8/Svneo cells in vitro. We used recombinant plasmid Rac1 shRNA to transfect HTR-8/Svneo cells with lipidosome to examine how Racl shRNA inhibit Racl activation of human extravillous trophoblasts HTR-8/SVneo. The HTR-8/SVneo cells were divided into four groups, namely, normal control (with only lipidosome), Cont-shRNA group (tranfect recombinant plasmid Cont-shRNA), Rac1 shRNA group (tranfect recombinant plasmid Rac1 shRNA) and β-catenin blocking group (providingβ-catenin inhibitor IWP-2). They were stably transfected in accordance with the optimization of transfection conditions provided by Regent Kit from Invitrogen. In addition, we utilized Western blot analysis to examine the activation and expression level of Racl. Western blot analysis was also performed to determine MMP9 and Snail protein expression in HTR-8/SVneo cells; Immunofluorescence was used to examine MMP9 expression; Transwell invasion assay was utilized to determine the invasiveness of HTR-8/SVneo cells and Real-time PCR was used to examine the expressions of Racl mRNA and MMP9 mRNA.ResultsThe GTPase pull down and Western blot analysis demonstrated that shRNA Racl recombinant plasmid expresses in HTR-8/SVneo cells and inhibits the expression of Racl gene. Western blot analysis suggested that β-catenin and Snail were expressed in HTR-8/SVneo cells in normal control and Cont-shRNA, and the expressions in Racl shRNA and P-catenin blocking were lower than in normal control and there were statistically significant differences (P<0.05). Immunofluorescence demonstrated that MMP9 was mainly expressed in HTR-8/SVneo cells and weaker staining of MMP9 was observed in Racl shRNA and β-catenin blocking, having significant differences. Furthermore, Western blot analysis and Real-time PCR proved that shRNA-Rac1 can significantly inhibit expressions of MMP9 protein and mRNA in HTR-8/SVneo cells (P<0.05). Transwell invasion assay found no significant differences in the number of penetrating cells between normal control and Cont-shRNA (P>0.05), whereas the number in Racl shRNA and P-catenin blocking was significantly lower than normal control, having significant differences (P<0.05).ConclusionsRac1 plays a role in Snail activation via eliciting the expression of P-catenin in HTR-8/SVneo cells and significantly increases the MMP9 expression and invasiveness of extravillous trophoblasts, suggesting that Racl signaling pathway may be involved in extravillous trophoblast invasion.