Study Of The Function Of MiR-10b And MiR-200c In Pre-eclampsia By Targeting SFlt-1

Pre-eclampsia(PE) is a leading cause of morbidity and mortality to maternity andfoetus worldwide. Insufficient utero placental oxygenation is believed to be responsiblefor the disease. However, what molecular events are involved in hypoxic responses andhow they affect placental development remain unclear.Recently many studies have found that soluble fms-like tyrsine kinase-1(sFlt-1)were highly overexpressed in pre-eclampsia maternal serum, which may be related to thepathogenesis and pathophysiology of pre-eclampsia．As to the mechanism of thepathogenesis of pre-eclampsia, it is currently believed that something caused abnormalincrease of sFlt-1in the starting early onset preeclampsia, leading to endothelial injuryvascular remolding and systemic capillary placenta. But the mechanism involved insFlt-1abnormality is not clear.MicroRNAs (miRNAs) are small noncoding RNAs molecules, which have beenproven to have a critical role in regulation of gene expression. Recent studies have shownthat some miRNAs are every highly expressed in human and mouse placenta, indicatingpost-transcriptional gene regulation may be associated with PE incidence. Using the microarray analysis, we have previously found that several miRNAs,including miR-10b and miR-200c, were down-regulated in placentas of patients withsevere PE compared with normal placentas. And we predicted that sFlt-1was a target ofmiR-10b and miR-200c by bioinformatics analysis. Therefore，it is necessary to study themolecular mechanism causing the abnormal expression of sFlt-1and the effect to thebiological function of sflt-1in PE placentas Our study may provide a new clue to thestudy in the aetiology of pre-eclampsia.Objective:1. To investigate the expression of miR-10b and miR-200c in the placenta betweensevere preeclampsia and the normal group, to validate whether there are relationshipbetween miR-10b or miR-200c and sFlt-1.2. To verify the targeting regulatory relationship between miR-10b\miR-200c andsFlt-1.3. To investigate the biological functions of miR-10b in the normal humantrophoblast cell lines,.Methods:1. ELISA analysis was performed to investigate the sFlt-1differential expression indifferent gestational maternal blood serum as well as the third trimester of severe PE(s-PE) in patients with normal pregnancies peripheral blood, quantitative real-time PCR(real-time PCR) analysis was performed to verify the expression of miR-10b andmiR-200c between severe PE group with normal pregnant group.2. Construct GV126-sFlt-1-3’UTR, which has a full length of3’UTR of sFlt-1, andGV126-sFlt-1-3’UTR–mut1， GV126-sFlt-1-3’UTR–mut2， which bearing fournucleotide substitution in the core binding sites. Dual-luciferase reporter system wasused to verify whether sFlt-1was a direct target of miR-10b/miR-200c RT-PCRanalysis was performed to verify the expression of miR-10b and sFlt-1mRNA betweenoverexpression group and normal group following over-expression of miR-10b. ELISA analysis was performed to verify the protein expression of sFlt-1between these twogroups.3. MTT, flow cytometry and invasion of small room, three-dimensional cell formingwere performed to detect the cell apoptosis, proliferation, cell cycle, infiltration and theVascular endothelial function in normal human trophoblast cell line (HTR-8/SVneo).Results:1. The expression of maternal serum sFlt-1was significantly higher in s-PE groupthan control. Real-time-PCR showed that the expression of miR-10b in the placenta ofs-PE was significantly lower （0.43±0.21VS1.08±0.32）(P＜0.05). However, theexpression of miR-200c was higher than normal group（5.26±0.35VS1.07±0.03）(P＜0.05), Both of them were correlated with maternal blood serum of sFlt-1expression.2. Bioinformatics analysis predicted that sFlt-1was a target of miR-10b andmiR-200c. Then, we constructed GV126-sFlt-1-3’UTR，which has a full length of sFlt-1mRNA3’UTR. GV126-sFlt-1-3’UTR-mut1(10b) and GV126-sFlt-1-3’UTR-mut2(200c), in which there were four nucleotides substitution in the core binding sites. Usingdual-luciferase reporter gene system, we observed a significant decrease (≈40%) ofluciferase activity in the presence of pre-miR-10b whereas there were no differences inluciferase activity in the presence of pre-miR-200c. These results confirmed that sFlt-1isa direct target of miR-10b. Furthermore, up-regulation of miR-10b in HTR-8/SVneocells led to the42%decrease of sFlt-1protein, whereas negative contral group didn’tcause sFlt-1protein change. The increased sFlt-1expression in miR-10b up-expressedcells was due to the mimcs of miR-10b.3. MTT, flow cytometry, cell proliferation and apoptosis assay results showed thatthere were no significant difference in experimental group and the control (P＞0.05) afterthe inhibiten of miR-10b. Cell transwell results showed that there was significantdifference between the experiments group and the control group. Infiltration capacity inexperiments group was increased compared to control group.(P＜0.05). Trophoblast cellculture medium decreased the forming of human vascular endothelial cells following inhibition of miR-10b.Conclusion:1. miR-10b and miR-200c are significantly differential expressed in pre-eclampticpregnancies placenta, suggesting the differential expression were related with theabnormal expression of sFlt-1, which was involved in the pathogenesis of pre-eclampsia.2. sFlt-1is a direct target of miR-10b, suggesting the potential role of miR-10b inthe etiology of PE through regulating sFlt-1.3. Through stimulating trophoblastic infiltration capacity and promoting the cells toproduce more sFlt-1, miR-10b affected vascular endothelial molding, reducedextravillous cytotrophoblast invasion, shallowed implantation of the placenta and finallyinvolved in the pathogenesis of pre-eclampsia.