The Role And Mechanism Of Long Non-Coding RNA MIR193BHG In The Pathogenesis Of Preeclampsia

BackgroundPreeclampsia(PE)is a pregnancy complication that seriously endangers the health of mother and child,mainly manifested as new-onset hypertension in pregnant women after 20 weeks of gestation,accompanied by one or more organ dysfunctions.The incidence of PE ranges from 2%-8%,and it is one of the main causes of high maternal and perinatal morbidity and mortality.The etiology and pathogenesis of PE have not been fully clarified so far.It is generally believed that the decreased infiltration ability of placental trophoblasts and the impediment of uterine spiral artery remodeling,resulting in "shallow implantation" of the placenta are the key links in the pathogenesis of PE.So far,no effective preventive and therapeutic measures have been found for PE.Termination of pregnancy is the only method that can prevent the progression of the disease,but it will lead to adverse pregnancy outcomes related to iatrogenic preterm birth.Therefore,looking for key regulatory molecules in the pathogenesis of PE,and in-depth study of their roles and molecular mechanisms in the pathogenesis of PE,is of great significance for elucidating the etiology of PE and realizing the early diagnosis and treatment of PE.Long non-coding RNAs(lncRNAs)are a class of RNA transcripts with no protein-coding capacity and lengths greater than 200 nucleotides.Studies have found that lncRNAs can regulate gene expression and function at the epigenetic,transcriptional and post-transcriptional levels,and participate in various biological processes such as cell differentiation,proliferation,apoptosis,and invasion.LncRNAs are closely related to the occurrence and development of various diseases,including tumors,diabetes,neurodegenerative diseases,and cardiovascular diseases.A variety of abnormally expressed lncRNAs have been found in the placental tissues,decidual tissues and peripheral blood of PE patients.However,the research on lncRNAs in PE is still at an early stage,and the functions and mechanisms of a large number of lncRNAs remain to be explored.Therefore,in this study,the differentially expressed lncRNAs in the placental tissues of PE patients and normal pregnant women were firstly screen out by lncRNA microarray technology.Then,the first trimester extravillous trophoblast cell line(HTR-8/SVneo)was used as an in vitro cell model to examine the effects of overexpression and knockdown of MIR193BHG on its biological behavior.Finally,RNA-sequencing(RNA-seq)technology was used to sequence the transcriptome of the HTR-8/SVneo cell line overexpressing MIR193BHG to screen and verify the downstream genes and signaling pathways that MIR193BHG may regulate,so as to preliminarily explore the role and mechanism of MIR193BHG in the pathogenesis of PE.This subject is mainly divided into the following three parts:Part Ⅰ Screening and validation of differentially expressed lncRNAs in placental tissues of preeclampsiaObjectives1.To screen out the differentially expressed lncRNAs in placental tissues of preeclampsia and normal pregnant women using lncRNA microarray technology.2.To screen out the lncRNA for follow-up research by validating candidate lncRNAs in expanded placental tissue samples and analyzing their clinical diagnostic value for preeclampsia.Methods1.The placental tissues of 30 pregnant women with early-onset preeclampsia who underwent cesarean delivered at the Third Affiliated Hospital of Zhengzhou University from May 2018 to July 2019 were collected as the PE group;30 cases of placental tissues from normal pregnant women collected during the same period were collected as the control group.2.The lncRNA microarray was used to detect the expression profiles of lncRNAs in 4 samples of PE group and 4 samples of control group.The abnormally expressed lncRNAs in the placental tissues of preeclampsia were screened out with the fold change>2 and P<0.05 as the screening criteria.3.The expression levels of MIR193BHG,PROX1-AS1 and GATA3-AS1 in 30 samples from PE group and 30 samples from control group were detected by real-time quantitative PCR(qRT-PCR).4.The clinical diagnostic value of MIR193BHG,PROX1-AS1 and GATA3-AS1 in placental tissues for preeclampsia was analyzed using Receiver Operating Characteristic Curve(ROC curve).Results1.The results of lncRNA microarray analysis showed that compared with the control group,there were 246 differentially expressed lncRNAs in the PE group(Fold Change>2,P<0.05),of which 159 were up-regulated and 87 were down-regulated.2.qRT-PCR results showed that the expression levels of MIR193BHG(P<0.001),PROX1-AS1(P<0.05)and GATA3-AS1(P<0.05)were significantly increased in the PE group compared with the control group.3.The results of ROC analysis showed that the area under the curve of MIR193BHG was 0.819(P<0.001,95%confidence interval:0.711-0.927),the area under the curve of GATA3-AS1 was 0.690(P<0.05,95%confidence interval:0.556-0.824),The area under the curve of PROX1-AS1 was 0.640(P>0.05,95%confidence interval:0.498-0.782).Among the three lncRNAs,MIR193BHG has the best clinical diagnostic value for preeclampsia.Brief summaries1.The lncRNA expression profile in placental tissues of patients with preeclampsia was significantly altered.2.The elevated expression level of MIR193BHG in placental tissues may be related to the pathogenesis of preeclampsia.Part Ⅱ Effects of MIR193BHG on biological behaviors of trophoblast cellsObjectivesTo study the effects of MIR193BHG on the proliferation,apoptosis,migration and invasion of trophoblast cells,and to further reveal the mechanism of MIR193BHG in the pathogenesis of preeclampsia.Methods1.The subcellular localization of MIR193BHG in HTR-8/SVneo cells was detected by RNA fluorescence in situ hybridization(RNA-FISH)and nucleocytoplasmic separation experiments.2.MIR193BHG Smart Silencer was used to transfect HTR-8/SVneo cells to construct a low-expressing cell model of MIR193BHG,and qRT-PCR technology was used to detect the knockdown efficiency.3.The MIR193BHG overexpression plasmid was used to transfect HTR-8/SVneo cells to construct the MIR193BHG overexpression cell model,and the overexpression efficiency was detected by qRT-PCR technology.4.The effects of knockdown and overexpression of MIR193BHG on the proliferation of HTR-8/SVneo cells were detected by CCK-8 assay.5.The effects of knockdown and overexpression of MIR193BHG on the migration of HTR-8/SVneo cells were detected by scratch assay.6.The effects of knockdown and overexpression of MIR193BHG on the invasion of HTR-8/SVneo cells were detected by Transwell assay.7.The effects of knockdown and overexpression of MIR193BHG on the apoptosis of HTR-8/SVneo cells were detected by flow cytometry using Annexin V-FITC/PI kit staining.Results1.The results of RNA-FISH and nucleocytoplasmic separation experiments showed that MIR193BHG was present in both the nucleus and cytoplasm,and was mainly distributed in the nucleus.2.The results of qRT-PCR showed that after HTR-8/SVneo cells were transfected with MIR193BHG Smart Silencer,the expression level of MIR193BHG was significantly lower than that in the control group(P<0.05);after HTR-8/SVneo cells were transfected with the MIR193BHG overexpression plasmid,the expression level of MIR193BHG was significantly higher than that in the control group(P<0.001).3.The results of CCK-8 experiment showed that the proliferation of HTR-8/SVneo cells in the MIR193BHG knockdown group was significantly higher than that in the control group at 24h,48h and 72h(P<0.05,0.01,0.01);the proliferation of HTR-8/SVneo cells in the MIR193BHG overexpression group was significantly lower than that in the control group at 24h,48h and 72h(P<0.05,0.001,0.01).4.The results of flow cytometry showed that the apoptosis rate of HTR-8/SVneo cells in the MIR193BHG knockdown group was significantly lower than that in the control group(P<0.05);the apoptosis rate of HTR-8/SVneo cells in the MIR193BHG overexpression group was significantly higher than that in the control group(P<0.001).5.The scratch assay results showed that there was no significant difference in the migration of HTR-8/SVneo cells between the MIR193BHG knockdown group and the control group at 24h after scratching(P>0.05);the migration of HTR-8/SVneo cells in the MIR193BHG knockdown group was significantly higher than that in the control group at 48h after scratching(P<0.05);the migration of HTR-8/SVneo cells in the MIR193BHG overexpression group was significantly lower than that in the control group at 24h and 48h after scratching(P<0.001,0.05).6.The results of Transwell invasion assay showed that the invasion ability of HTR-8/SVneo cells in the MIR193BHG knockdown group was significantly higher than that in the control group(P<0.05);the invasion ability of HTR-8/SVneo cells in the MIR193BHG overexpression group was significantly lower than that in the control group(P<0.05).Brief summariesMIR193BHG may participate in the occurrence and development of preeclampsia by inhibiting the proliferation,migration and invasion of trophoblast cells,and promoting apoptosis of trophoblast cells.Part III:Exploration of the mechanism of MIR193BHG affecting the biological behaviors of trophoblast cellsObjectives1.To explore the downstream molecules and signaling pathways regulated by MIR193BHG,and to further explore the pathogenesis of MIR193BHG involved in preeclampsia.2.To verify whether MIR193BHG can affect biological behaviors of trophoblast cells such as proliferation,apoptosis,migration and invasion through the p53 pathway at the cellular level.Methods1.RNA-seq technology was used to detect the mRNA expression profile of HTR-8/SVneo cells in the MIR193BHG overexpression group and the control group,and the differentially expressed mRNAs were screened with |logFC|>0.5,P<0.05 as the screening criteria.2.For differentially expressed mRNAs,KEGG pathway enrichment and protein-protein interaction network construction were performed simultaneously to screen out the downstream molecules and signaling pathways regulated by MIR193BHG.3.The expression levels of p53 mRNA and protein in HTR-8/SVneo cells after overexpression of MIR193BHG were detected by qRT-PCR and Western Blot.4.HTR-8/SVneo cells were transfected with p53 small interfering RNA(siRNA),and the expression levels of p53 mRNA and protein were detected by qRT-PCR and Western Blot techniques.5.HTR-8/SVneo cells were divided into three groups:co-transfected with MIR193BHG overexpression plasmid and p53-siRNA group(MIR193BHG-OE+p53-siRNA),transfected with MIR193BHG overexpression plasmid and siRNA negative control group(MIR193BHG-OE+NC-siRNA),transfected with empty plasmid and siRNA negative control group(NC-OE+NC-siRNA).CCK-8,flow cytometry,scratch assay and Transwell assay were used to detect the proliferation,apoptosis,migration and invasion of cells in each group.Results1.Based on the RNA-seq results,a total of 147 differentially expressed genes were screened(|logFC|>0.5,P<0.05),of which 63 were up-regulated and 84 were down-regulated in the MIR193BHG overexpression group.2.The KEGG pathway enrichment analysis of differentially expressed genes showed that the differentially expressed genes were mainly enriched in cellular senescence,cell cycle,fluid shear stress and atherosclerosis,and p53 signaling pathway.The protein-protein interaction network showed that TP53 was the hub gene in the network.3.The results of qRT-PCR showed that the relative expression level of p53 mRNA in the MIR193BHG overexpression group was significantly higher than that in the control group(P<0.05).Western Blot results showed that the relative expression level of p53 protein in the MIR193BHG overexpression group was significantly higher than that in the control group(P<0.05).4.Compared with the negative control group,the expression levels of p53 mRNA and protein in HTR-8/SVneo cells were significantly down-regulated after transfection with p53-siRNA(P<0.001).The knockdown efficiency of p53-siRNA-3 was relatively high and was used for subsequent experiments.5.After co-transfection of MIR193BHG overexpression plasmid and p53-siRNA,compared with the(NC-OE+NC-siRNA)group,the proliferation of HTR-8/SVneo cells in(MIR193BHG-OE+NC-siRNA)group was significantly decreased at 24h,48h and 72h(P<0.05,0.001,0.001);Compared with the(MIR193BHG-OE+NC-siRNA)group,there was no significant difference in the proliferation of HTR-8/SVneo cells in(MIR193BHG-OE+p53-siRNA)group at 24h(P>0.05),but significantly increased at 48h and 72h(P<0.05,0.05).Compared with the(NC-OE+NC-siRNA)group,the apoptosis rate of HTR-8/SVneo cells in(MIR193BHG-OE+NC-siRNA)group was significantly increased(P<0.01);Compared with the(MIR193BHG-OE+NC-siRNA)group,the apoptosis rate of HTR-8/SVneo cells in(MIR193BHG-OE+p53-siRNA)group was significantly decreased(P<0.05).Compared with the(NC-OE+NC-siRNA)group,the migration and invasion of HTR-8/SVneo cells in(MIR193BHG-OE+NC-siRNA)group were significantly decreased(P<0.01,0.01);Compared with the(MIR193BHG-OE+NC-siRNA)group,the migration and invasion of HTR-8/SVneo cells in(MIR193BHG-OE+p53-siRNA)group were significantly increased(P<0.05,0.05).Brief summariesMIR193BHG may regulate the proliferation,apoptosis,migration and invasion of trophoblast cells through the p53 pathway,thereby participating in the pathogenesis of preeclampsia.Conclusions1.The elevated expression of IncRNA MIR193BHG in placental tissues may be associated with the pathogenesis of preeclampsia.2.LncRNA MIR193BHG may participate in the pathogenesis of preeclampsia by inhibiting the proliferation,migration and invasion,and promoting the apoptosis of trophoblast cells through the p53 pathway.