MiR-145-5p-mediated Repression Of Cyr61 Is Involved In The Anti-invasive Effect Of TNF-α On HTR-8/SVneo Trophoblast Cells

Objective:Preeclampsia(PE)is a serious gestational disease and one of the leading causes of maternal mortality.The cause of PE has remained unclear,which is leading to poor treatment.Therefore,it is important to fully understand the pathogenesis of PE for effective treatment of this disease.Superficial trophoblastic cell infiltration outside the chorionic membrane is the main cause of preeclampsia.Cell migration and invasion ability are affected by a variety of molecules and signaling pathways.Screening the molecules that affect the occurrence and development of PE will help us to further understand the pathogenesis of PE.Human chorionic trophoblast cell lines outside HTR8/SVneo cells derived from early pregnancy trophoblastic cells,which have the chorionic trophocyte characteristics.HTR8/SVneo cells are widely applied in gestational disease(including PE)research.The purpose of this study is to investigate the effects and molecular mechanisms of TNF-α/miR-145-5p/ Cyr61 axis on HTR8/SVneo cells invasion and migration.Attempts to characterizeHTR8/SVneo cells migration and invasion will potentially have broad-reaching significance for PE treatment.Methods:(1)HTR8/SVneo cells were treated with different concentrations of growth factor beta(TGF-β),lysophosphatide acid(LPA),interleukin 35(IL-35),and tumor necrosis factor α(TNF-α)for 24 h,respectively.Cells migration was tested by wound healing assay.The aim of the above experiments was to screen a cytokine that has an effect on HTR8/SVneo cells invasion and migration.(2)According to the screening results,TNF-α was selected for further study to screen the downstream molecules of TNF-α that affect the migration ability of HTR8/SVneo cells,and different concentrations of TNF-α were used to treat HTR8/SVneo cells.Real time RT-PCR and Western blotting were used to detect the expression of invasion related transcription factor MSH homologous box protein 2(MSX2),FOXM1(FOXM1),FOXO1(FOXO1),signal transduction and transcription activator 3(STAT3),and cysteine-rich protein 61(Cyr61)at mRNA and protein levels,respectively.(3)According to the screening results,Cyr61 was identified as the main downstream molecule of TNF-α affecting the migration ability of HTR8/SVneo cells.Gene cloning technology was used to construct the expression vector of Cyr61 and the expression vector of Cyr61 targeting shRNA to transfect HTR8/SVneo cells.(4)To further confirm and explore the molecular mechanism of TNF-α affecting Cyr61,the expression profile of miRNAs was changed after TNF-α treatment of HTR8/SVneo cells by bioinformatics and real time RT-PCR,and miR-145-5p was detected to be increased after TNF-α treatment of HTR8/SVneo cells.Bioinformatics and dual luciferin reporting system were used to verify the inhibitory effect of miR-145-5p on Cyr61 mRNA.The transfected HTR8/SVneo cells weretransfected with synthetic miR-145-5p mimics and antagmiar,and the migration ability of the transfected cells was detected by wound healing assay and Transwell.Results(1)After 24 h treatment of HTR8/SVneo cells with different concentrations of TGF-?,LPA,IL-35 and TNF-α,the wound healing assay results showed that TGF-?,LPA and IL-35 had no significant effect on the migration ability of cells,but TNF-α with final concentrations of 10 ng/ml and 100 ng/ml could significantly inhibit the invasion ability of cells(P < 0.05).(2)Real time RT-PCR results showed that there was no significant change in the expression of invasion related transcription factors Msx2,FOXO1,FOXM1,STAT3 and Cyr61 mRNA in TNF-α-treated HTR8/SVneo cells.Western blotting results showed that there were no significant changes in the expression of invasion related transcription factors Msx2,FOXO1,FOXM1 and STAT3 in TNF-α-treated HTR8/SVneo,but the expression level of Cyr61 protein was significantly reduced(P<0.05).(3)The expression plasmid of Cyr61(pCDH-CMV-MCS-EF1-GFP-puro-Cyr61)and the expression plasmid of Cyr61 targeting shRNA(PLKO.1-Cyr61-shRNA-1 and PLKO.1-Cyr61-shRNA-2)were successfully constructed.The results of real time RT-PCR and Western blotting showed that the mRNA and protein levels of Cyr61 were significantly enhanced in the overexpressed cell lines(P < 0.05).The mRNA and protein levels of Cyr61 were significantly decreased in both knockdown cell lines(P <0.05),and pLKO.1-Cyr61-shRNA-1 was more effective than pLKO.1-Cyr61-shRNA-2.Therefore,we performed follow-up experiments with pLKO.1-Cyr61-shRNA-2 stable cells.Scratch test results showed that overexpression of Cyr61 could promote the invasion rate of HTR8/SVneo cells(P < 0.05),while knockdown of Cyr61 could significantly inhibit the invasion rate of HTR8/SVneo cells(P < 0.05).In the Transwell invasion experiment,we obtained similar results.Scratch assay and Transwell invasion assay also showed that HTR8/SVneo cells overexpressing Cyr61 had a significant turnover effect on TNF-α-induced invasion inhibition compared with control cells transfected with the vector.(4)Real time RT-PCR results showed that compared with the control group,the expression of miR-145-5p in TNF-α treated HTR8/SVneo cells was significantly increased(P<0.05).Using bioinformatics methods analysis,according to the results of Cyr61-3′-UTR with miR-145-5p has a combination sequence.Dual luciferase report system test results show the miR-145-5p mimics can combine Cyr61-3 ′-UTR and suppress gene expression;After the transfection of miR-145-5p mimics,the real time RT-PCR results showed that the expression of miR-145-5p was approximately 83 times higher than the original.Western blotting results showed that in the cells transfected with miR-145-5p mimics,the expression of Cyr61 decreased significantly,only about 26% of the original level.Scratch test and Transwell test results showed that transfection of miR-145-5p mimics in HTR8/SVneo cells could significantly inhibit the invasion rate of HTR8/SVneo cells(P<0.05),while transfection of miR-145-5p inhibitor could significantly enhance the invasion rate of HTR8/SVneo cells(P < 0.05).The scratch test results and Transwell test results after 24 h transfection with miR-145-5p inhibitor into HTR8/SVneo cells showed that miR-145-5p inhibitor could increase the invasion rate of HTR8/SVneo cells induced by 10 ng/ml TNF-α(P < 0.05).ConclusionTNF-α up-regulated the expression of miR-145-5p,and miR-145-5p overexpression decreased the expression of Cyr61 protein.miR-145-5p caninhibit the invasion of HTR8/SVneo cells.Cyr61 can reverse the inhibitory effects of TNF-α on the invasion of HTR8/SVneo cells.TNF-α inhibited the invasion of HTR8/SVneo cells by up-regulating miR-145-5p and then targeting the expression of Cyr61.