The Functional And Mechanical Research Of Circular RNA CircFAM 120A In Regulating The Decidualization Process Of Endometrial Stromal Cells

Background:recurrent implantation failure(RIF)is defined as patients who have undergone at least 3 fresh or frozen cycles but still failed to accomplish a clinical pregnancy after 4-6 high-scored cleavage stage embryos transfers or at least 3 good-quality blastocyst transfers.This is a great burden for patients both in economics and mental health.RIF is a burning problem that limits the pregnancy rate in assisted reproduction,and thus demands prompt solution.The pathogenesis of RIF is still unrevealed.Some people think the endometrial receptivity is one of the key factors that influence the outcome of embryo implantation.However,the past researches failed to find a specific molecule or histological characteristics as a biomarker to reliably evaluate the endometrial receptivity.Circular RNA(circRNA)is a group of non-coding RNA with a covalently closed circular structure.There may be various biological functions related to circRNA.And the mostly studied one is that it can participate in the development and progression of many diseases acting as a microRNA(miRNA)sponge to regulate expression of most mRNA and thus influence a various of biological functions such as cell proliferation,differentiation and apoptosis.The special circular structure is relatively stable and often show tissue and developmental stage specificity.circRNA has the potency to act as a molecule biomarker.At present,the researches relating cirRNA and endometrial receptivity are at an early stage.A study found some differentially expressed circRNA in the endometrium of implantation window period from RIF patients and non-RIF controls.But the concrete function or mechanism of how circRNA impacting the endometrium of RIF patients is still unclear.Objective:screen the circRNA that may participate in regulating the decidualization process of endometrium in the period of implantation window and study the potential downstream miRNA and mRNA.Deeper research on circRNA participating in the pathological process and molecular mechanism of RIF.Materials and methods:using Arraystar Human circRNA Array V2 to screen the differentially expressed circRNAs in the endometrium tissue which is in the period of implantation window from 10 controls and 8 RIF patients.And then using Quantitative Real-time PCR(qRT-PCR)to verify the expression of the selected circRNAs in an expanded sample size of 10:21.According to the relative expression level,fold changes between the two sample groups and bioinformatics analysis,circFAM120A was chose for further verification in experiment of cell functions.After downregulating the expression of circFAM120A in human endometrial stromal cells(hESCs)using small interfering RNA(siRNA),we analyzed its effect on the proliferation and/or decidualization of hESCs.In order to further understand biological characteristics of circFAM120A,we performed RNase R digestion,Fluorescent In Situ Hybridization(FISH)and RNA Binding Protein Immunoprecipitation(RIP).After downregulating the expression of circFAM120A in human endometrial stromal cells,we did RNA-seq in day 2 of proliferation and day 4 of decidualization.And we used qRT-PCR to verify the expression level of the mRNAs in endometrium tissues from 14 controls and 12 RIF patients.According to the relative expression level,fold changes between the two sample groups and bioinformatics analysis,TMEM245 and ABHD5 was chose as potential downstream mRNAs for further verification.We used siRNA to downregulate the expression of TMEM245 and ABHD5 in hESCs and analyzed their impact on proliferation and/or decidualization of hESCs.miR-29 was predicted as the mediator of circFAM120A influencing the expression of ABHD5 by bioinformatics analysis.We used luciferase reporter assay to confirm the combination mechanism of miR-29 and ABHD5.We performed the transfection of miR-29 mimics into hESCs to confirm the regulatory function of miR-29 on ABHD5 and the influence of miR-29 on decidualization.After suppressing circFAM120A in hESCs,we transferred miR-29 inhibitor to perform a rescue experiment to further verify the mechanism.Results:we screened 6 downregulated circRNAs from the endometrium of implantation window period from the array results.According to the verification from a larger sample size,we found the expression of circFAM120A downregulated significantly in the endometrium from RIF patients and the tendency is correspondence with the results from the array.PRL and IGFBP1,the marker genes of decidualization,was restrained significantly after we suppress the expression of circFAM 120A in human endometrial stromal cells.By EDU experiment,we discovered an increased proliferation positive cell ratio after downregulating circFAM 120A(p<0.05).Through flow cytometry,we found a significant decrease in G1 phase cells(p<0.05),but an obvious increase in S phase and G2 phase cells after the suppression of circFAM 120A(p<0.05,p<0.05).From the RNA-seq results,the expression of TMEM245 and ABHD5 were significantly inhibited after downregulating circFAM 120A.We verified in endometrium tissues and found the expression of TMEM245 and ABHD5 were significantly lower in endometrium of implantation period from RIF patients.After downregulating TMEM245 in hESCs and inducing decidualization,the decidualization marker genes PRL and IGFBP1 were both inhibited in day 4(p<0.05,p<0.05).EDU experiment indicated an increased proliferation positive cell ratio after downregulating TMEM245(p<0.0001).Through flow cytometry,we found a significant decrease in G1 phase cells(p<0.05),but an obvious increase in S phase(p<0.05).No statistically difference was found in G2 phase cells after the suppression of TMEM245.We also found that the decidualization marker genes PRL and IGFBP 1 were significantly suppressed after we knockdown ABHD5 or transferred miR-29 mimics in hESCs.Luciferase Reporter Assay indicated that the mechanism of miR-29 downregulating ABHD5 may through combining with the 3’UTR region of ABHD5.Using miR-29 inhibitor can partially rescue the phenotype of decidualization suppression by si-circFAM120A.Conclusion:downregulated circFAM 120A in the endometrium of implantation period of RIF patients may inhibit the expression of TMEM245 and ABHD5,thus influence the decidualization process of endometrial stromal cells.circFAM 120A may act as miR-29 sponge to regulate the expression of ABHD5,influencing embryo implantation and cause implantation failure.