Expression Of MicroRNAs And Their Related Functions In The Ovaries From Rat Model Of Polycystic Ovary Syndrome

Polycystic ovary syndrome(PCOS) is a complex and heterogeneous endocrine disorder, which is characterized by hyperandrogenemia, hyperinsulinemia, insulin resistance and chronic anovulation. PCOS patients present diversity of clinical manifestations, its short-term and long-term complications seriously affect the patients’ physical and mental health. PCOS is reported to be responsible for 75% of cases of anovulatory infertility and associated with a significantly increased risk of type 2 diabetes, cardiovascular disease and endometrial cancer. It follows that PCOS, which causes serious damage to women’s health, has become the focus of gynecologic endocrine experts. Now, the possible causes of PCOS include dysfunction of hypothalamics—pituitary—gonadal axis, hyperleydigism, insulin resistance, cell apoptosis and abnormal follicular development. The standpoint of cell apoptosis and abnormal follicular development contribute to the pathogenesis of PCOS attracted more and more attention from scholars, however, the underlying mechanisms need further studies.Micro RNAs(mi RNAs) are a class of endogenous single-stranded and small non-coding RNAs, about 18-24 nucleotide long and negatively regulate the expression of target genes at the post-transcriptional level by binding to the 3’untranslated region(UTR) of target m RNAs. Mi RNAs are highly expressed in the uterus, fallopian tubes, ovaries and the other female reproductive organs, and participate in follicular maturation,fertilization, implantation and early embryonic development. Dysfunctional mi RNAs were implicated in the pathogenesis of many diseases, previous studies demonstrated that mi RNAs were differently expressed in follicle granulosa cells of PCOS patients, and abnormal expression of some mi RNAs(let-7a, let-7i and mi R-92b) might be associated with apoptosis of ovarian granulosa cells. It indicated that mi RNAs perhaps contributed to apoptosis of ovarian granulosa cells, which regulated the pathogeneisis of PCOS. In this study, we aim to explore the key mi RNAs and their regulatory mechanism related to the development of PCOS.Due to the samples from PCOS patients were difficult to obtain, we established rat model of PCOS. With the help of mi RNAs deep sequencing technologies, real-time PCR, Western Blot, gene transfection and dual-luciferase reporter gene assay, we tried to find the key mi RNAs and their downstream pathways participating in the pathogenesis of PCOS.1. Establishment of rat model of PCOSWe used letrozole lavage method to establish the rat model of PCOS. The rats in model group were administrated with letrozole dissolved in 1% carboxymethylcellulose at the dose of 1 mg/kg for 23 days. The rats in control group were administered with carboxymethyl cellulose at the same dose for 23 days. It was obvious that control rats showed regular estrus cycles, whereas model rats showed disrupted estrus cycles during 13 days of evaluation. HE staining showed pathological changes of ovaries in model rats, including more atretic follicles, fewer corpus luteums and fewer growing ovarian follicles. Furthermore, ELISA showed that plasma levels of LH、FSH and T were dramatically increased while plasma level of E2 was dramatically decreased in model rats. Taken together, these results demonstrated that we have successfully established the rat model of PCOS.2. Deep sequencing and bioinformatics analysisWe extracted total RNA from ovarian tissue and employed deep sequencing to screen differential expression of mi RNAs in model rats and control rats. Deep sequencing showed that a total of 129 mi RNAs were differentially expressed in the ovaries from model rats compared to the controls, including 49 mi RNAs upregulated and 80 mi RNAs downregulated. To validate the results of deep sequencing, we selected 4 differentially expressed mi RNAs(mi R-201-5p, mi R-34b-5p, mi R-141-3p and mi R-200a-3p) for real time PCR analysis. Compared to control group, mi R-34b-5p, mi R-141-3p and mi R-200a-3p displayed significantly decreased expression while mi R-201-5p displayed significantly increased expression in model rats. These results showed that the expression of all 4 mi RNAs analyzed by real time PCR were in concordance with the deep sequencing data. Bioinformatic analysis revealed that these 4 mi RNAs were predicted to target 2060 genes with different functions. GO and Pathway analysis supported that the mi RNAs regulate oocyte meiosis, MAPK signaling, PI3K-Akt signaling, Rap1 signaling, Notch signaling, reproductive process and cell killing.3. Functional identification of mi R-141-3pMi R-141-3p displayed significantly decreased expression in model ovaries. MTT assay showed that cell viability was dramatically increased when mi R-141-3p was overexpressed, but was decreased when mi R-141-3p was interfered. Flow cytometry showed that cell apoptotic rate was dramatically decreased when mi R-141-3p was overexpressed, but was increased when mi R-141-3p was interfered. Bioinformatics analysis predicted that DAPK1 might be the target gene of mi R-141-3p because the results showed that the 3’-UTR region of DAPK1(death associated protein kinase 1) had sequences complementary with mi RNA-141-3p. Real-time PCR results showed that compared with the control rats, mi R-141-3p was dramatically decreased and DAPK1 was dramatically increased in ovaries of model rats, which indicated their expression were significantly negative correlation. After transfection of mi R-141-3p mimics and inhibitor to granulosa cells, both DAPK1 m RNA and protein were negatively correlated with mi R-141-3p. Dual-luciferase reporter gene assay showed that the luciferase activity of mi R-141-3p mimics group was significantly decreased compared to control group when transfecting Wt-DAPK1 3’-UTR luciferase reporter vector to HEK 293 T cells;the luciferase activity of mi R-141-3p mimics group and mi R-mimics NC group had no significant difference when transfecting Mut-DAPK1 3’-UTR luciferase reporter vector to HEK 293 T cells. These results established that DAPK1 was the target gene of mi R-141-3p because mi R-141-3p could bind to DAPK1 3’-UTR and negatively regulate DAPK1 transcription.These data indicate that differentially expressed mi RNAs are probably involved in the etiology of PCOS. Decreased mi R-141-3p promote cell apoptosis through targeting DAPK1, further leading to the development of PCOS. Our findings will contribute to identify mi RNA-141-3p/DAPK1 as novel diagnostic markers and therapeutic targets for PCOS.