Function And Mechanism Studies Of CircSTK40 In Low Endometrial Receptivity Contributing To Recurrent Implantation Failure

Background:RIF is generally defined as the failure to achieve a clinical pregnancy after more than three transfers of high-quality embryos or multiple transfers of at least 10 embryos,incurring large financial losses and considerable mental stress to these patients.The etiology of RIF is clinical challenging involving embryo,uterine,tubal,and Immunological factors.However,the pathogenesis remains to be elucidated in most cases.Endometrial receptivity was proposed as a measurement of the ability of endometrium to allow an embryo to attach,invade,migrate,and finally embed in the deeper layer during the window of implantation.The establishment of endometrial receptivity is primarily coordinated by ovarian estrogen and progesterone and requires an elaborate interplay of different transcription factors,cytokines,and signaling pathways.Some benign gynecological disorders are the well-known clinical causes for implantation failure,progesterone resistance and dysregulation of some molecules,including leukemia inhibitory factor,αvβ3 integrin,and HOXA10,have been found in these disorders and result in the impairment of endometrial receptivity.Although many of receptivity-associated proteins have been described,their interactions and regulatory factors in the network of endometrial development are still not fully understood,especially in the pathogenesis of unexplained RIF.Circular RNAs（circRNAs）are an evolutionarily conserved class of stable ncRNAs with covalently closed ends,which are widely expressed in a tissue-specific and cell-specific manner.CircRNAs have been reported to perform a broad array of biological functions,for example,by acting as sponges for microRNAs sponges or proteins,by binding with proteins to modulate their interactions and activity,or by encoding peptides,etc.Increasing evidence has implicated circRNAs in the pathogenesis of different diseases,such as neurological disorders,cardiovascular diseases,diabetes mellitus,and many types of cancers.Recent studies demonstrated a potentially central role for circRNAs in endometrial receptivity.In humans,although variations in circRNA expression have been reported among patients with RIF,the functions of the differentially expressed circRNAs,as well as other molecular mechanisms potentially involved in implantation failure have yet to be determined.Objective:To investigate the circRNA expression profile in luteal-phase endometrium from RIF patients and to further explore the functions and underlying mechanisms of differently expressed circRNAs in the process of endometrial receptivity establishment,to clarify the pathogenesis of RIF from the epigenetic angle and providing a foundation for the development of targeted therapies for RIF.Methods:CircRNA expression profile was detected on the luteal-phase endometrial samples from 8 vs.8 age-matched control and RIF women using Arraystar Human circRNA Array V2.Based on the cut-off values of fold change>2 and P<0.05,several abundant,differentially expressed circRNAs were validated with divergent primers and RT-qPCR in the luteal-phase endometrium from an independent cohort of 16 control and 17 RIF women.RNase R experiment and Sanger sequencing were performed to verify circSTK40 as a circular RNA.Cell fractionation,cytoplasm/nucleus fractionation,and fluorescence in situ hybridization（FISH）assay were performed to investigate the distribution of circSTK40.After circSTK40 overexpression in both the THESC cell line and primary hESCs,in vitro induction of decidualization,TUNEL assay during oxidative stress,Western blots,EdU assay,and cell cycle assay were perfomed to explore the role of circSTK40 in decidualization process,cell apoptosis and proliferation.For further mechanism study,we performed transcriptome sequencing and RNA pull-down assay combined with mass spectrometry（MS）analysis to detect the differentially expressed genes after circSTK40 overexpression and the proteins interacted with circSTK40.Subsequent assays of RNA immunoprecipitation（RIP）and co-immunoprecipitation were performed to detect the interactions between RNA and proteins,or between different proteins,and to investigate the signal pathways involved.Finally,rescue experiments using the specific inhibitors MK2206 for AKT and 17AAG for HSP90 were performed to confirm the molecular mechanisms for cell phenotypesResults:Among 1436 differently expressed circRNAs detected with circRNA array,17 differentially expressed circRNAs were validated in an independent cohort of 16 control and 17 RIF women.In particular,circSTK40（circBase accession:hsa_circ₀011692）,a circular RNA generated by back splicing from exon 4 and 5 of the STK40 gene,was the most abundant of the significantly up-regulated circRNAs in RIF endometrium.We determined that circSTK40 had greater resistance to RNase R than the linear STK40 mRNA,and that circSTK40 was both localized in both the cytoplasm and nucleus of endometrial stromal cells（ESCs）using cell fractionation,cytoplasm/nucleus fractionation and FISH.The results of in vitro induction of decidualization,TUNEL during oxidative stress,Western blots,EdU and cell cycle demonstated that circSTK40 overexpression in ESCs hindered the decidualization process as indicated by a decrease of the mRNA levels of the decidualization markers prolactin（PRL）and insulin-like growth factor binding protein 1（IGFBP1）;inhibited cell apoptosis as indicated by a decreased number of TUNEL-positive cells during oxidative stress and an increase in the BCL2/BAX ratio;and inhibited cell proliferation as indicated by a decreased number of EdU-positive cells and S-phase cells.Additionally,we also found that circSTK40 was significantly up-regulated in ESCs under oxidative stress.Assays of RNA pull-down combined with MS and RIP showed that circSTK40 directly interacted with HSP90 and CLU.CircSTK40 overexpression resulted in significant up-regulation of HSP90 protein levels and didn’t alter HSP90 mRNA levels.After CHX-mediated inhibition of protein synthesis,we found that circSTK40 overexpression retarded the degradation of HSP90 and prolonged its half-life,and treatment with MG132 to inhibit proteasomal degradation led to stabilization of HSP90 protein levels.Co-IP assays showed that circSTK40 interfered in HSP90 interactions with HSP90 potential E3 ligase RBBP6,and interactions with CLU which regulates the proteasomal degradation of targeted proteins.In agreement with our results showing the effects of circSTK40 overexpression,we found that knockdown of CLU with siRNA interference disrupted the interactions between HSP90 and RBBP6,and further attenuated the degradation of HSP90 after CHX-mediated protein synthesis inhibition in ESCs.CircSTK40 overexpression and the resulting high levels of HSP90 enhanced HSP90-pAKT interaction,activated the AKT pathway,leading to a decrease in FOXO1.Notably,lower protein levels of FOXO1 were also identified by Western blot analysis in RIF endometrium compared to that in the control group.Rescue experiments using MK2206 and 17AAG showed that the reduction of FOXOl in circSTK40-overexpressing ESCs was significantly mitigated,and circSTK40 overexpression-mediated decreases in apoptosis and decidualization were also abolished by MK2206 and 17AAG.Interestingly,MK2206 and 17AAG within a certain range of doses resulted in a significant increase in decidualization,as indicated by a dose-dependent increase in mRNA levels of PRL and IGFBP1.Using transcriptome sequencing and RT-qPCR,we found that circSTK40 overexpression reduced the mRNA and protein levels of BMP2,and lower mRNA levels of BMP2 were also identified in RIF endometrium compared to that in the control group.Pull-down assay demonstrated that circSTK40 potentially interacts with TCF/LEF family.In agreement with our results showing the effects of circSTK40 overexpression,we found that knockdown of BMP2 with siRNA interference inhibited decidualization levels and cell proliferation.Conclusions:CircSTK40 hinders the decidualization process,inhibits cell apoptosis and proliferation via modulating HSP90/AKT/FOXO1 axis and regulating BMP2 expression levels,resulting in low endometrial receptivity which contributing to the pathogenesis of RIF.Although overexpression of circSTK40 impairs endometrial receptivity,it also enhances ESC survival during stress conditions.