| ObjectivePreeclampsia is a serious pregnancy disorder affecting both maternal and fetal health.However,the pathogenesis of preeclampsia has not been fully understood.This study aimed to investigate the key microRNAs(miRNAs)in the development of preeclampsia,to explore its mechanism in the occurrence of preeclampsia and to find a new target for the pathophysiology of preeclampsia.Materials and Methods1.High-throughput sequencing and Differential expression analysis of miRNAsDuring the cesarean section,placental tissues were collected within 5 minute of delivery of the placenta.A high-throughput miRNA sequencing analysis for the placental tissues from patients with preeclampsia and controls was conducted,followed by investigation of differentially expressed miRNAs(DEMs)and functional enrichment analysis.MiR-200b-3p was selected as the key DEM of preeclampsia by CCK8 and Transwell migration pre-assay.Next,we verified the expression of miR-200b-3p in placental tissues of the two groups was confirmed to be different by qPCR.2.The function of miR-200b-3pTo further investigate the function of miR-200b-3p in preeclampsia development,miR-200b-3p was overexpressed in HTR8 cells by transfection,and their negative control(NC)using Lipofectamine 2000.After 48 h of transfection,cells were harvested for the subsequent experiments.2.1 CCK8 assay(Cell Counting Kit-8)After transfection,HTR8 cells(2000 cells)were seeded onto 96-well plates.At indicated time points(0,24,48,72,and 96h),10 μl of CCK-8 reagent was added to each well and continued to incubate the cells for 1.5 h at 37℃.The optical density(OD)value of each well was detected with a microplate reader.2.2 Transwell migration assay The cells(6×104)at the logarithmic growth stage in different groups were Trypsin digested,suspended in serum-free media and then seeded into the upper Transwell inserts.The 600 μL medium containing 30%FBS was then added into the lower Transwell inserts.After 24 hours of culture,the cells were stained,imaged and counted using a fluorescent microscope。2.3 Scratch wound healing assay The HTR8 cells(5 ×105 cells/well)were plated in culture dishes.Cells were cultured overnight and formed a confluent monolayer.Then,a linear scratch wound was created in a straight line with a pipet tip.Afterwards,the serum-free media were added to culture cells for 24 h at 37℃ in a humidified incubator with 5%CO2.At 0 and 24 h after scratch,wound closure was photographed and analyzed.2.4 Flow cytometry for detection of cell apoptosis Cell apoptosis of different groups were detected by the FITC-Annexin V Apoptosis Detection Kit(BD Biosciences)using flow cytometry.Briefly,HTR8 cells in different groups were starved in serum-free medium for 24 h.Cell(1 ×106 cells/ml)were then harvested,followed by double staining with 5 μl FITC-Annexin V and 10 μl Propidium iodide(PI).After incubation at room temperature and in dark for 5 min,the percentage of apoptotic cells was detected with a flow cytometry and analyzed by Flowjo software.3.Based on GSE177049 and miRWalk 2.0 tool,PFN2 were obtained which was regarded as the target genes of miR-200b-3p in the development of preeclampsia.The expression of PFN2 in miR-200b-3p overexpression group and control group was verified by Western blot.Dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-200b-3p and profilin 2(PFN2).Finally,Transwell migration assay and scratch wound healing assay were used to verify whether overexpression of PFN2 reversed the inhibitory effect of Mir-200b-3p on trophoblast migration.Results1.A total of 12 DEMs including miR-200b-3p were identified between preeclampsia placental tissues and control placental tissues,which were significantly enriched in several pathways,such as cell adhesion molecules(CAMs)and tight junction.Among these,seven miRNAs namely hsa-miR-200b-3p,hsa-miR-708-5p,hsa-miR-10b-5p,hsa-miR-549a-5p,hsa-miR-124-3p,hsa-miR-2113,and hsa-miR-200a-3p,were significantly upregulated in the preeclampsia samples,while the other five miRNAs,including novel-m0267-5p,novel-m0224-3p,hsa-miR-2682-3p,hsa-miR-137-3p,and hsa-miR-4664-5p,were remarkably downregulated.We verified the expression of miR-200b-3p by qPCR.The results showed that miR-200b-3p expression was significantly upregulated in the placental tissues of patients with preeclampsia compared to that in the placental tissues of controls(P<0.01),in line with the result of miRNA sequencing analysis.2.To further investigate the function of miR-200b-3p in preeclampsia development,miR-200b-3p was overexpressed in HTR8 cells by transfection.The cell proliferation was monitored by CCK-8 assay,and the results indicated that compared with NC group,overexpression of miR-200b-3p remarkably decreased the cell viability of HTR-8 cells after 48 h of transfection(P<0.05).Moreover,both Transwell migration assay and scratch wound healing assay revealed that overexpression of miR-200b-3p significantly inhibited the migration of HTR-8 cells(P<0.01).Furthermore,the results of flow cytometry revealed that overexpression of miR-200b-3p dramatically promoted the apoptosis of HTR-8 cells(P<0.001).3.The target gene of miR-200b-3p was investigated based on gene expression profile GSE177049 and miRWalk 2.0 database.The target relationship between miR-200b-3p and profilin 2(PFN2)was investigated in vitro.The results showed that the expression level of PFN2 protein was significantly down-regulated in HTR-8 cells after overexpression of miR-200b-3p(P<0.05).The results of luciferase activity assay showed that only the luciferase activity of PFN2-WT was significantly inhibited after co-transfection with miR-200b-3p(P<0.05).To further investigate whether miR-200b-3p contribute to preeclampsia via targeting PFN2,we overexpressed the expression of PFN2 by transfection.The results of qPCR and western blot assay showed that the expression levels of PFN2 mRNA and protein were all significantly increased after transfection(P<0.001),suggesting the high transfection efficiency.Furthermore,both Transwell migration assay and scratch wound healing assay revealed that the inhibitory effect of miR-200b-3p overexpression on the migration of HTR-8 cells was significantly reversed after overexpression of miR-200b-3p and PFN2 at the same time(all P<0.01).ConclusionsOur findings reveal that miR-200b-3p is upregulated in the placental tissues from patients with preeclampsia.The effects of miR-200b-3p overexpression on the proliferation,migration,and apoptosis of HTR8 trophoblast cells were investigated in vitro.The results indicated that overexpression of miR-200b-3p suppressed cell proliferation and migration but promoted apoptosis of trophoblast cells.PFN2 was confirmed as a target of miR-200b-3p,and overexpression of PFN2 reversed the inhibitory effects of miR-200b-3p overexpression on trophoblast cell migration.Our findings reveal that miR-200b-3p is upregulated in the placental tissues from patients with preeclampsia and promotes preeclampsia development via PFN2.miR-200b-3p may serve as a promising therapeutic target against preeclampsia.. |
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