Wei Peng1,2,3, Ying Liu1,2,3, Hongbo Qi1,2,3, Qingshu Li4,5,6. 1. Department of Obstetrics, The First Affiliated Hospital of Chongqing Medical University, 400016, Chongqing, China. 2. Chongqing Key Laboratory of Maternal and Fetal Medicine, Chongqing Medical University, 400016, Chongqing, China. 3. Joint International Research Laboratory of Reproduction and Development of Chinese Ministry of Education, Chongqing Medical University, 400016, Chongqing, China. 4. Chongqing Key Laboratory of Maternal and Fetal Medicine, Chongqing Medical University, 400016, Chongqing, China. qingshuli77@163.com. 5. Joint International Research Laboratory of Reproduction and Development of Chinese Ministry of Education, Chongqing Medical University, 400016, Chongqing, China. qingshuli77@163.com. 6. Department of Pathology, School of Basic Medicine, Chongqing Medical University, 1 Yixueyuan Rd, Yuzhong District, 400016, Chongqing, China. qingshuli77@163.com.
Abstract
BACKGROUND: Proper differentiation of trophoblasts in the human placenta is essential for a successful pregnancy, whereas abnormal regulation of this process may lead to adverse pregnancy outcomes, especially preeclampsia (PE). However, the underlying mechanism of trophoblast differentiation remains unclear. Previous studies have reported the involvement of alpha-actinin-4 (ACTN4) in the actin cytoskeleton dynamics and motility. Hence, we hypothesized that ACTN4 may act as an important regulator in the normal proliferation and differentiation of trophoblasts during early pregnancy. METHOD: To test this hypothesis, we collected villous tissues from women undergoing a legal pregnancy termination during 6-10 weeks of gestation and explanted them for cell culture and siRNA transfection. We also obtained placental tissues from PE patients and healthy pregnant women and isolated the primary cytotrophoblast (CTB) cells. The expression of ACTN4 in the CTBs of placental villi and during the differentiation of CTBs into STBs was detected by immunofluorescence, immunohistochemistry (IHC), and EdU proliferation assays. Besides, villous explant, Matrigel invasion, transwell migration assay, and Wound-healing assay were performed to identify the possible role of ACTN4 in the outgrowth of explants and the invasion, migration, and proliferation of cell column trophoblasts (CCTs). Western blot analysis was carried out to compare the protein expression level of AKT, Snail activities, and epithelial-to-mesenchymal transition (EMT) in the villi or HTR8/SVneo cells with ACTN4 knockdown. RESULTS: ACTN4 was highly expressed in CTB cells and interstitial extravillous trophoblast (iEVT) cells but not found in the syncytiotrophoblast (STB) cells in the first trimester villi. Downregulation of ACTN4 led to reduced trophoblast proliferation and explant outgrowth ex vivo, as well as iEVT invasion and migration in vitro due to disrupt of actin filaments organization. Such ACTN4 inhibition also decreased AKT and Snail activities and further impeded the EMT process. In addition, ACTN4 expression was found to be downregulated in the iEVTs from preeclamptic placentas. CONCLUSIONS: Our findings suggest that ACTN4 may act as an important regulator of trophoblast proliferation and differentiation during early pregnancy, and dysregulation of this protein may contribute to preeclampsia pathogenesis.
BACKGROUND: Proper differentiation of trophoblasts in the human placenta is essential for a successful pregnancy, whereas abnormal regulation of this process may lead to adverse pregnancy outcomes, especially preeclampsia (PE). However, the underlying mechanism of trophoblast differentiation remains unclear. Previous studies have reported the involvement of alpha-actinin-4 (ACTN4) in the actin cytoskeleton dynamics and motility. Hence, we hypothesized that ACTN4 may act as an important regulator in the normal proliferation and differentiation of trophoblasts during early pregnancy. METHOD: To test this hypothesis, we collected villous tissues from women undergoing a legal pregnancy termination during 6-10 weeks of gestation and explanted them for cell culture and siRNA transfection. We also obtained placental tissues from PE patients and healthy pregnant women and isolated the primary cytotrophoblast (CTB) cells. The expression of ACTN4 in the CTBs of placental villi and during the differentiation of CTBs into STBs was detected by immunofluorescence, immunohistochemistry (IHC), and EdU proliferation assays. Besides, villous explant, Matrigel invasion, transwell migration assay, and Wound-healing assay were performed to identify the possible role of ACTN4 in the outgrowth of explants and the invasion, migration, and proliferation of cell column trophoblasts (CCTs). Western blot analysis was carried out to compare the protein expression level of AKT, Snail activities, and epithelial-to-mesenchymal transition (EMT) in the villi or HTR8/SVneo cells with ACTN4 knockdown. RESULTS:ACTN4 was highly expressed in CTB cells and interstitial extravillous trophoblast (iEVT) cells but not found in the syncytiotrophoblast (STB) cells in the first trimester villi. Downregulation of ACTN4 led to reduced trophoblast proliferation and explant outgrowth ex vivo, as well as iEVT invasion and migration in vitro due to disrupt of actin filaments organization. Such ACTN4 inhibition also decreased AKT and Snail activities and further impeded the EMT process. In addition, ACTN4 expression was found to be downregulated in the iEVTs from preeclamptic placentas. CONCLUSIONS: Our findings suggest that ACTN4 may act as an important regulator of trophoblast proliferation and differentiation during early pregnancy, and dysregulation of this protein may contribute to preeclampsia pathogenesis.
Entities:
Keywords:
ACTN4; Invasion and migration; Preeclampsia; Proliferation; Trophoblast