The Role And Mechanism Of Enhancer RNA In Unexplained Recurrent Spontaneous Abortion

The loss of pregnancy before 20 weeks or fetal weight less than 500g for 2 or more times in clinical practice refer to recurrent spontaneous abortion(RSA).There are still 50%to 60%of the RSA can not find a clear cause in clinical,this is called unexplained recurrent miscarriage(URSA).At present,the specific pathogenesis of URSA is still not clear,known causes include genetic factors,immune factors,endocrine disorders,anatomical abnormalities,infection factors,etc..In recent years,epigenetics and its related research has become a frontier field of life sciences.As an important part of epigenetic regulation,long non-coding RNA(IncRNA)has important biological functions.Studies have shown that IncRNA plays a very important role in the X chromosome inactivation,gene imprinting,HOX gene cluster regulation and the embryonic development process Some lncRNAs can be transcribed from the genome of the enhancer elements,which are called enhancer RNA(eRNA).Although some studies have confirmed that eRNA plays an important regulatory role in embryonic development,there is no report on the role of eRNAs in URSA.Therefore,it is urgent and important to make clear the expression pattern,expression regulation mechanism and function of eRNA in vitro or in vivo.In this study,high-throughput sequencing was used as the starting point to screen eRNAs associated with URSA,and qRT-PCR verification of independent populations was used to identify sequencing data.And the in vitro cell test can further explore the role of eRNA in URSA and provide the basis.Part I Screening and validation of enhancer RNA for unexplained recurrent spontaneous abortionThe RNA transcribed from the enhancer sequence is the enhancer RNA(eRNA).There is no report on the role of eRNAs in URSA,so screening for URSA-related eRNAs by sequencing is particularly important.In this part of study,high-throughput sequencing was used as the starting point to screen eRNAs associated with URSA,and qRT-PCR verification of independent populations was also used.Through RNA-seq,we found 131 different IncRNA in URSA and normal control villi.By comparing the ChIP-Seq data of human embryonic stem cell histone modified with ENCODE(the validated enhancer region),38 potential functional eRNA were screened.KEGG and GO analyses of 38 potentially functional eRNA associated with unexplained recurrent spontaneous abortions were shown to be concentrated in inflammation and infection related pathways,then 12 eRNA which may be involved in inflammation and infection related pathways were selected for independent population verification,and the results were consistent with the sequencing results.Among them,Inc-SLC4A1-1 participates in the most inflammatory and infectious related pathways.It provide the basis to explore the role of eRNA in URSA.Part ?The role and mechanism of enhancer RNA lnc-slc4al-1 in unexplained recurrent spontaneous abortionSuccessful pregnancy is inseparable from the normal functioning of trophoblast cells.Therefore,by studying the functional changes of trophoblast cells in the maternal-fetal interface,the mechanism of various pathological pregnancy can be further explored.In this part,the human trophoblast cell HTR-8/SVneo cell line overexpressed in Inc-SLC4A1-1 were used.The effects of overexpression of lnc-SLC4A1-1 on trophoblast cell function including cell proliferation,cell migration,cell apoptosis,cell cycle were observed by CCK-8,Transewell and flow cytometry.The role of lnc-SLC4A1-1 in URSA was preliminarily explored by detecting the changes of downstream target genes after Overexpression of lnc-SLC4A1-1.Lnc-slc4a1-1 has effect on the proliferation,migration,cycle and apoptosis of trophoblast cells,which indicates the role of Inc-slc4a1-1 in regulating trophoblast function.ChIP-qPCR were used toconfirm that lnc-slc4a1-1 was an enhancer RNA,overexpression of lnc-SLC4A1-1 resulted in up-regulation of TLR4 expression and increased TNF-? and IL-1? concentrations.After using TLR4 antagonist,it was found that compared with verexpression of Inc-SLC4A1-1 alone,the concentration of IL-1?decreased(P<0.05).Therefore,Inc-SLC4A1-1 may promote the expression of TLR4 by recruiting H3K27ac and activating the Toll-like receptor signaling pathway.TLR4 activates downstream signal molecules TNF-?,IL-1?,causes inflammation and loses the advantage of Th2,leading to URSA happened.