The Expression Profiling Of MRNA,lncRNA And MiRNA In The Endometrium Of Pcos Mice During The Receptive Phase

Background and ObjectivePolycystic ovary syndrome(PCOS)is a common reproductive endocrine and metabolic disorder with heterogeneous clinical manifestation.The typical symptoms include oligomenorrhea or amenorrhea,ovulatory dysfunction,polycystic ovarian morphological changes and hyperandrogenism,which can also be accompanied by abnormalities such as infertility,obesity,insulin resistance and lipid metabolism alterations.Studies have shown that women with PCOS have an increased risk of adverse pregnancy outcomes such as early pregnancy loss,gestational hypertension,gestational diabetes,and premature delivery.The dysfunction of the endometrium plays a vital role in the occurring of adverse pregnancy outcomes.The growth and differentiation of endometrial tissue are regulated by a variety of hormones and factors,predominantly estrogen and progesterone.The abnormal endocrine states such as excessive androgen and insulin resistance in PCOS women can affect the microenvironment and receptivity of the endometrium,thus hindering the normal establishment of pregnancy.In recent years,extensive studies have focused on the role of non-coding RNAs in various biological processes,and many studies have been conducted on non-coding RNAs related to endometrial receptivity.Non-coding RNAs regulates the physiological and pathological changes of the genome expression of the endometrium.Long noncoding RNAs(lncRNAs)and micro RNAs(miRNAs)are two types of noncoding RNAs that regulate gene expression.lncRNAs are greater than 200 nt in length,which can regulate gene expression at multiple levels such as transcription level and post-transcription level.Studies have shown that endometrial lncRNAs can regulate the signaling pathways associated with sex hormone to regulate endometrial receptivity.miRNA is a type of small noncoding RNA with a length of about 22 nt,which can bind to mRNA in a base complementary pairing manner,thereby inhibiting transcript translation or degrading target genes.Studies have shown that the expression of classic endometrial markers such as LIF and pinopode is regulated by miRNA.At present,studies have detected and analyzed the endometrial transcripts of PCOS,but as far as we know,there is no research reporting on the expression profiling of endometrial non-coding RNA of PCOS during the implantation window.In this study,PCOS mouse model was constructed by injecting dehydroepiandrosterone(DHEA)and RNA-seq was used to acquire the RNA expression profiling of endometrial tissue in PCOS mice during the implantation window.Moreover,differentially expressed mRNAs,lncRNAs,and miRNAs in the endometrium of PCOS mice were screened out and further bioinformatic analysis was carried out to find out the key molecules that may be related to endometrial receptivity.This study may provide a basis for exploring the regulation mechanism of the effects which lncRNAs and miRNAs have on the PCOS endometrial receptivity.Materials and methods1.The PCOS mouse model was constructed by the subcutaneous injection of DHEA.The methods of model identification include monitoring the estrus cycle through vaginal smears,calculating the changes of mouse body weight,measuring the blood testosterone levels,and observing the morphological changes of ovarian tissue under a microscope.2.Vasectomy was performed on sexually mature male mice(2 months old)and the male infertility state was checked about half a month after the surgery,after which they were caged with the female mice from PCOS group and control group.Endometrial tissues(n=4)was obtained from the mice on the day 4.5(the time when the vaginal plug was seen=day 0.5),the endometrial total RNA was extracted and RNA-seq was performed subsequently.3.After obtaining the sequencing data of mRNAs,lncRNAs and miRNAs of the endometrium,differentially expressed RNAs were selected through analysis.Gene ontology(GO)and KEGG pathway enrichment analysis of differentially expressed genes and predicted target genes was performed and a competing endogenous(ceRNA)network was established by R and Cytoscape software.Results1.The PCOS mice lost their original regularity of the estrus cycle and stayed in estrus stage.Through the observation of the ovarian tissue slices under the microscope,it was seen that PCOS ovaries contained more expansive cystic follicles,accompanied by decreased layers of granulosa cells.PCOS mice gained weight faster(P<0.05),and their serum testosterone levels were higher(P<0.05).2.There were 399 differentially expressed mRNAs in the endometrium of PCOS mice,174 were up-regulated,and 225 were down-regulated.Differentially expressed genes were enriched in GO Terms such as adipocyte differentiation,fatty acid metabolism,reproduction process and hormone metabolism process.KEGG pathway enrichment results included JAK-STAT signal pathway and PPAR signal transduction pathway,and differentially expressed genes included Adipoq and Lep which were related to PCOS metabolism.3.A total of 401 lncRNAs were screened as differentially expressed lncRNAs,220 were up-regulated,and 181 were down-regulated.3025 cis target genes were predicted,and the KEGG pathway enrichment analysis indicated that JAK-STAT signal pathway and NK cell-mediated cytotoxicity were involved.There are 18 lncRNA-mRNA coexpression pairs.4.There were 16 differentially expressed miRNAs in the PCOS mice endometrium,and8 of them were up-regulated.The predicted 1385 target genes were mainly enriched in the calcium signaling pathway,cAMP signaling pathway,Wnt signaling pathway and insulin secretion.62 lncRNAs were predicted to have interactions with miR-107-3p,miR-331-3p,miR-466d-5p and miR-1941-5p,of which 36 lncRNAs were not differentially expressed,and the other 26 lncRNAs have no detectable expression.Moreover,there were 23 genes that were predicted to target miR-466d-5p,miR-331-3p and miR-3069-5p,and that were also differentially expressed in endometrium.5.A ceRNA regulatory network was constructed using miR-466d-5p and miR-331-3p as the bridges.The nodes in the network consisted of 23 mRNAs and 24 lncRNAs.Conclusion1.The expression profiling of mRNA、lncRNA and miRNA changed in PCOS mice endometrium during the receptive phase.2.Fatty acid metabolism,NK cell mediated cytotoxicity,insulin secretion,JAK-STAT and PPAR signaling pathways may be involved in the regulation of alterations in PCOS endometrial receptivity.