Functional And Mechanism Study Of MiR-146b-5p In Early Unexplained Miscarriage

Backgroud Early miscarriage,also known as early spontaneous abortion and defined as an intrauterine pregnancy loss of an embryo or fetus before 12 weeks of gestation,is the most common complication of human pregnancy.Despite various systematic examinations,nearly 50% of cases fail to identify an etiology,which is termed early unexplained miscarriage(UM).According to recurrence or not,UM can be divided into unexplained sporadic miscarriage(USM)and unexplained recurrent spontaneous abortion(URSA).URSA refers to two or more consecutive losses of ultrasound or histologically confirmed UM with the same spouse,while the first or inconsecutive loss of UM is referred to as USM.However,UM usually without any clinical symptoms,lacks reliable biomarkers and effective treatments.Therefore,it is urgent to investigate its pathogenesis and explore new biomarkers and intervention methods for clinical treatments.It has been shown that micro RNAs(miRNAs)are differentially expressed in villus tissue,decidua and peripheral blood of UM compared to normal pregnancy(NP),which may be involved in the pathogenesis of UM,thus showing promise as UM biomarkers and therapeutic targets.Our previously sequenced data showed that miR-146b-5p was significantly upregulated in UM villus tissues.Importantly,it has been demonstrated that miR-146b-5p is highly conserved among species and also upregulated in the plasma and serum of pregnant women with URSA.It can negatively regulate immune and inflammatory diseases,and also acts as an oncogene or tumor suppressor gene to regulate tumor growth.The appropriate inflammatory response is extremely important for embryo implantation.Extravillous trophoblast cells(EVT),which play a critical role in embryonic implantation and the establishment of a maternal-fetal relationship,own biological characteristics similar to cancer cells.Given these,we speculate that miR-146b-5p may be involved in the pathogenesis of UM.However,its role and related mechanisms have not been fully investigated in UM,therefore,further studies are necessary.Objective We detect the expression and evaluate the diagnostic potential of miR-146b-5p in villous tissue of UM,USM and URSA on the basis of previous transcriptome sequencing;Its effects on mouse placental development were further investigated via a animal model;Finally,Its function and related mechanism were investigated in extravillous trophoblast cell line HTR-8/SVneo to explore its pathophysiological mechanism in UM.Methods1.Villus tissue samples from UM(n=52),including USM(n=32)and URSA(n=20),were collected.Villus tissue samples from matched normal pregnancy(NP,n=52)acted as controls.RT-q PCR was conducted to compare the expression of miR-146b-5p between UM,USM,URSA and NP,and ROC curves were performed to evaluate its diagnostic potential.2.On E5.5,NC agomir(left)and mmu-miR-146b-5p agomir(right)were locally injected into the uterine cavity of ICR pregnant mice,respectively.On E13.5,mice were sacrificed to observe the development of embryos in the uterus,measure wet weight of the uterus,and calculate the embryo resorption rate.mmumiR-146b-5p expression in placental tissues was detected by RT-q PCR.3.Transfection of mimics and inhibitor to overexpress or suppress miR-146b-5p in HTR-8/SVneo cells.Effects of miR-146b-5p on cell proliferation,cycle,migration,invasion and endothelial‐like network formation ability were examined via CCK-8,Ed U,flow cytometry,scratch assay,transwell assay and tube formation assays,respectively.Additionally,associated protein levels were detected by western blot.4.HTR-8/SVneo cells after overexpression of miR-146b-5p were sent for m RNA sequencing.Downregulated differential expressed genes(DEGs)were intersected with Targetscan 7.2 to predict potential target genes,which were further validated by RT-q PCR,western blot,Ago2 RIP-PCR and dual luciferase assay.5.IRAK1 overexpressed cells were constructed by lentiviral infection in HTR-8/SVneo,in addition,si-IRAK1 was transfected to knock down the expression of IRAK1.Effects of IRAK1 on cell proliferation,cycle,migration and invasion were observed by CCK-8,Ed U,flow cytometry,scratch assay,and transwell migration and invasion assays.Moreover,related protein changes were detected by western blot.miR-146b-5p mimics were transiently transfected into IRAK1-overexpressed cells,then,Ed U and transwell migration and invasion assays were used to observe whether upregulation of IRAK1 could rescue the inhibition of trophoblast function by miR-146b-5p.Results1.Compared with NP,miR-146b-5p was significantly upregulated and displayed good diagnostic potential in UM,USM and URSA.2.Intrauterine local injection of mmu-miR-146b-5p agomir could upregulate the expression of miR-146b-5p in mouse placenta,reduce wet uterine weight and increase embryo resorption rate.3.HTR-8/SVneo cells after overexpression of miR-146b-5p showed attenuated proliferation,migration and invasion,increased G1 / G0 phase cells and decreased S phase cells;Cyclin E and cyclin B1,MMP9 and vimentin proteins were significantly downregulated,while E-cadherin was markedly upregulated;However,there was no significant change in the ability of endothelial-like network formation.By contrast,HTR-8/SVneo cells with miR-146b-5p inhibitor showed enhanced proliferation,migration and invasion abilities.4.Sequenced data identified a total of 1297 differentially expressed genes(DEGs),with 611 upregulated and 686 downregulated.Some immune inflammation-related factors,such as CXCL1,CXCL8,CXCL12 and IL1 B,were significantly downregulated.DEGs were enriched in focal adhesion,thyroid hormone and TGF-β signaling pathways and involved in regulating biological processes such as cell migration,growth and apoptosis.The downregulated differential genes were intersected with Targetscan 7.2 to obtain 37 potential target genes,and 10 target genes related to proliferation,migration and invasion were validated by RT-q PCR,and their expression were consistent with RNA-seq,with IRAK1 and ADAM19 showing the most significant changes.After overexpression of miRNA-146b-5p,the protein expressions of IRAK1 and ADAM19 were both decreased;miR-146b-5p,IRAK1 m RNA and ADAM19 m RNA were all enriched on Ago2 protein.miR-146b-5p mimics could bind to the3’UTR region of IRAK1 m RNA and ADAM19 m RNA,respectively,to inhibit luciferase activity.5.HTR-8/SVneo cells after overexpression of IRAK1 showed increased proliferation,migration and invasion,decreased G1 / G0 phase cells and increased S phase cells;Cyclin D,Cyclin E and MMP9 protein expressions were upregulated;In contrast,knockdown of IRAK1 inhibited cell proliferation,migration and invasion;increased G1 / G0 phase cells and decreased S phase cells.Cyclin D,cyclin E and MMP9 protein expression was downregulated.Overexpression of IRAK1 could partially rescue the suppression of miR-146b-5p mimics on the proliferation,migration and invasion of HTR-8/SVneo cells.Conclusions Elevated miR-146b-5p in villous tissue is closely associated with the pathogenesis of UM,and it is a causative factor and potential biomarker of UM.miR-146b-5p suppresses EVT cells proliferation,migration,invasion,cell cycle progression and inflammatory response of embryo implantation by downregulating IRAK1 and ADAM19,resulting in insufficient EVT cell plugs to block spiral arteries,together with diminished ability to anchor the uterine decidua and disorder of remodeling uterine spiral arteries,and ultimately villous tissue dysplasia,which is involved in the pathogenesis of UM.Downregulation of miR-146b-5p expression ameliorates EVT function and may be a biomarker and potential therapeutic target in UM.