Research On Identification Of IncRNA CD36-005 Expression In The Uteri Of PCOS Rat Model And Related Functions

Background:Polycystic ovary syndrome?PCOS?is one of the most common and complex endocrine disorders in women of reproductive age.The clinical features of PCOS are highly heterogeneous.Patients with PCOS are commonly characterized by persistent ovulatory disorder,ovarian polycystic morphology and hyperandrogenism.In addition,in women with PCOS,the risk of adverse endometrial outcomes including decreased endometrial receptivity,endometrial hyperplasia and endometrial cancer increase.The decreased endometrial receptivity of patients with PCOS is mainly due to the endocrine disorder,insulin resistance and ovulation induction therapy.Recent years,the molecular mechanism of endometrial receptivity has become the focus of reproductive medicine researches.Long non-coding RNAs?lncRNAs?are defined as a class of non-coding transcripts with the length more than 200 nucleotides.Although lncRNAs lack the capacity to code for proteins,they can regulate gene expression at epigenetic,transcriptional,posttranscriptional,and other levels.Dysfunctional lncRNAs contribute to the pathogenesis of many human diseases and the role of lncRNAs in the pathogenesis of several endometrial diseases has also been reported in recent studies,including implantation failure or spontaneous miscarriage,endometrial hyperplasia,adenomyosis,endometriosis,and endometrial cancer.However,we knew little about the role of lncRNAs in the pathogenesis of adverse endometrial outcomes of PCOS.Methods and Results:1.Identification of lncRNA CD36-005 expression in the uteri of PCOS rat modelThe PCOS rat model was induced by continuous intragastric administration of letrozole?1 mg/kg/d?for 23 days.The results showed that the control group and the model group were generally in good condition;the weight of the rats in model group was significantly higher than that in normal control group?P<0.05?;the estrus cycle of the rats in model group was irregular and showed continuous dioestrus;the concentration of serum T,LH and FSH of the rats in model group was increased while the concentration of E₂ was decreased?P<0.05?;HE staining of ovarian tissue showed typical polycystic changes in the ovarian morphology of the rats in model group;HE staining of uterus showed abnormal morphological changes including thinner endometrial stromal layer and reduced uterine gland in the uterine morphology of the rats in model group.The above results indicate that the PCOS rat model was successfully constructed.Result of the RT-PCR analyses showed that the expression level of lncRNA CD36–005 in the uteri of PCOS rat model was significantly higher than that in the normal?P<0.05?.2.Identification of mRNAs related to endometrium function regulated by lncRNACD36–005 in rat endometrial stromal cellsWe used primary endometrial stromal cells from rat uteri as the in-vitro model,and performed RNA sequencing?RNA-seq?technology and bioinformatic analyses after lncRNA CD36–005 overexpression to investigate its potential role from a more comprehensive perspective.We also conducted CCK-8 assay to determine the effect of lncRNA CD36–005 on the proliferation activity of stromal cells,which is the first step of decidualization.A total of 55 target genes of lncRNA CD36–005 were identified?|Log₂Fold Change|?1,P<0.05?.Among the 55 differentially expressed mRNAs,28 mRNAs were upregulated and 27 mRNAs were downregulated in the Ad-CD36–005 group.Bioinformatics analyses showed that these differentially expressed genes were associated with diverse signaling pathways,including cell growth and death,transport and catabolism,signal transduction,lipid metabolism and so on.According to references and our interest,nine mRNAs from the results of RNA-seq were selected for further validation by using RT-PCR analysis,including Hmgn5,Nr5a2,Dll4,Nos2,Clip3,Wbp1,Brms1,Entpd1 and Fam50a.The results showed that the RT-PCR results of expression levels of all nine mRNAs confirmed were consistent with the RNA-seq data.3.The role of lncRNA CD36-005 and Rsad2 in endometrial decidualizationThe expression level of lncRNA CD36-005 and its downstream target gene Rsad2 in the uteri on days 5-8 of pregnancy rats were significantly decreased?P<0.05?,and in the uteri on days 6-8 of pregnancy,the expression levels of lncRNA CD36-005 and Rsad2 in the implantation site were significantly lower than those in the non-implantation site?P<0.05?.The lower expression of lncRNA CD36-005 and Rsad2 were also detected in artificially induced decidualization in vivo and stromal cells under in vitro decidualization?P<0.05?.After overexpression of lncRNA CD36-005 in pregnancy rats,the size of embryos decreased significantly and the expression level of Rsad2 was also significantly increased in the uteri infected with Ad-CD36-005?P<0.05?.Meanwhile,overexpression of lncRNA CD36-005 could reduce the expression of Prl8a2 and Hand2 which were two markers for decidualization,whereas inhibition of lncRNA CD36-005 with specific siRNA could enhance their expression?P<0.05?.Inhibition of Rsad2 with specific siRNA could enhance their expression of Prl8a2?P<0.05?,while the expression of Hand2 was not significantly changed.These results suggest that lncRNA CD36-005 may be involved in the regulation of decidualization of endometrial stromal cells by targeting Rsad2,thereby inhibiting the process of endometrial decidualization.Conclusions:This study identified the dysregulated expression level of lncRNA CD36-005 in the uteri of PCOS rat model and provided a list of potential target mRNA genes of lncRNA CD36–005 in endometrial stromal cells.Further studies on the molecular function and mechanism showed lncRNA CD36–005 may inhibit the endometrial decidualization by targeting Rsad2.