The Study Of Let-7b On The Biological Functions Of Trophoblast Cell And Its Role In Preeclampsia

Objective: Preeclampsia(PE)is a pregnancy-induced disease.So far,the underlying pathogenesis has not been fully elucidated.The pathological changes of preeclampsia started from the excessive autophagy and apoptosis of placental trophoblast,and the decreased invasion ability,which eventually led to the "shallow implantation" of placenta and the dysfunction of placental vascular remodeling.An increasing number of studies has reported that Let-7b plays an important role in various cellular processes and diseases,such as inflammation,infection,osteogenic differentiation,and cancers,which is involved by regulating the functions of inflammation,differentiation,apoptosis,autophagy and invasion of these cells.We speculate that let-7b may participate in the regulation function of placental trophoblast cell,such as apoptosis,autophagy,EMT and invasion and eventually participate in the occurrence of PE.Methods:1.Detection of Let-7b expression level in PE model of trophoblast cells and Human Preeclamptic placenta Tissues1.1 The expression level of let-7b in PE model of trophoblast cells and the control group were detected by qRT-PCR.1.2 The expression level of Let-7b in normal(n=19)and PE(n=21)(diagnosed as severe PE)placental tissues was detected by qRT-PCR.1.3 The expression levels of extracellular regulated protein kinases(ERK)in PE placenta and PE model of trophoblast cells were detected by western blot.2.Effects of let-7b on trophoblast biological functionHuman trophoblast HTR-8/SVneo cells were transduced with control or let-7b over-expressing lentivirus.The expression levels of let-7b was detected by qRT-PCR.2.1 Cell proliferation was measured by the CCK-8 and EdU assays.2.2 Apoptosis was measured by flow cytometry assay and Annexin V/DAPI staining,The expressions of apoptosis-associated proteins caspase3 were measured by western blot.2.3 The expressions of autophagy-associated proteins LC3 B were measured bywestern blot,autophagy-associated genes levels were measured by qRT-PCR.2.4 To explore whether the Let-7b expression also affects trophoblast inflammation,we assessed inflammatory cytokine expression in trophoblasts,Production of TNF?,IL-6,and IL-10 was assessed by qRT-PCR.2.5 Cell invasion were assessed by using Transwell,Expression of key proteins associated with invasion and EMT marker E-cadherin and Vimentin were measured by Western blot analysis.3.Regulation mechanism of Let-7b on EMT and invasion of trophoblastits3.1 Validation of Let-7b for ERK pathway activation/inhibition and invasion mechanisms for related PathwayHuman trophoblast HTR-8/SVneo cells were transduced with control or let-7b over-expressing lentivirus.In order to elucidate the signaling cascade,ERK were detected by western blot.The ERK kinase inhibitors U-0126 were probed to explore the possible regulatory mechanism of let-7b on EMT and invasion in HTR-8/SVneo cells.3.2 Screening and detection of Let-7b downstream mRNAThe public online database of Targetscan,miRBase were searched to select the potential role of specific Let-7b that have a consensus binding site in the 3'-untranslated region(3'-UTR)of TGFBR1.The let-7b overexpressed trophoblast cells and the corresponding negative controls were used to detect the the levels of TGFBR1 protein.3.3 Studying the mechanism of let-7b on regulating ERK pathway through theregulation of downstream TGFBR1In order to explore the regulation mechanism of let-7b on ERK signaling pathway and the mechanism of invasion in trophoblast cells,the level of TGFBR1 in let-7b overexpressed trophoblast cells was detected.Deep investigate the regulatory relationship and regulation mechanism of upstream Let-7b and target proteins TGFBR1 and downstream ERK signaling pathway.Results:1.expression of let-7b was significantly down-regulated compared with the normal control in PE model of trophoblast cells.We found that Let-7b was significantlydownregulated in Human PE placental Tissues.Meanwhile,The expression levels of extracellular regulated protein kinases(ERK)in PE placenta and PE model of trophoblast cells were decreased.2.Let-7b over-expression in HTR-8/SVneo cells significantly promoted cell proliferation and significantly inhibited cell apoptosis and autophagy.Meanwile,Let-7b overexpression resulted in significantly decreased TNF-? production,but increased IL-6production by trophoblasts.At the same time,let-7b was found to increase the EMT process of trophoblast cells and promote trophoblast invasion.3.Let-7b can activated the ERK pathways,In addition,ERK pathways participate Let-7b-mediated increase in invasion and EMT of trophoblast cells,Let-7b activates the ERK signalling pathway in a TGFBR1-mediated manner in trophoblast cells.Conclusions: Through this study,It is found that the expression of let-7b decreased in PE model of trophoblast cells,Let-7b may be involved in PE pathogenesis by regulating the EMT and invasive function of trophoblast cells,Meanwhile,Let-7b may also participate in proliferation? apoptosis ?autophagy of trophoblast cell and immunity of maternal-fetal interface,Let-7b/TGFBR1/ERK pathways was probably involved in the pathogenesis of PE.All these findings may contribute to our knowledge regarding PE progression and to the development of a novel therapy target for PE.