Characterization Of MiRNA-483-5p And Targeted Gene ERK1 In Theregulation Of Proliferation-apoptosis Balance Of Granulosa Cells And Development Of PCOS

【Background】 Polycystic ovary syndrome(PCOS) is the most common reproductive endocrine disorder in childbearing-aged women, characterized with polycystic ovaries, hyperandrogenism, insulin resistance and chronic anovulation. Cumulus granulosa cells surrounding the oocyte are involved in different aspects of PCOS pathology. Several studies suggested that mi RNAs play an important regulatory role at the post-transcriptional level in cumulus granulosa cells. As important regulatory molecules in eukaryocytes, micro RNAs(mi RNA) participate in female reproductive process widely including follicle and oocyte maturity, narmal fertilization and early embro development.【Objective】Our objective was to describe the altered mi RNA expression profiles and mi RNA targeted signaling pathways in PCOS.【Methods】(1) The restropective analysis of clinical cases. All the samples used in this study were collected from the Centre for Reproductive Medicine of Anhui Provincial Hospital Affiliated with Anhui Medical University between January 2013 and March 2014. Case–control study that involved 21 women with PCOS(PCOS group) and 20 women without the disease(Controls group). All these patients were undergoing ICSI-ET with a standard long stimulation protocol due to male factor infertility. The age, the body mass index, the bases five hormones, serum glucose(Glu) concentration, serum insulin(INS) concentration were compared, respectively, between two groups.(2) The laboratory investigation of miRNA in GCs. 1) The collection of cumulus cells experimental study group and experimental control group 2) With a new generation of sequencing technology(NGS) Illumina Hiseq 2000 and CPSS tool to analyze the deep sequencing and data for small RNA. 3) Detecting the expression of mi RNA duing the two groups of granulosa cell, and tcompare the differences between them. 4) Thenforecast the differenct expression of mi RNA downstream target genes, and use the Gene Ontology(GO) and KEGG Pathway analysis software for its potential downstream regulatory pathway enrichment and screening. 5) Last, through of regulatory pathway of real-time quantitative RT-PCR, Western Blot in patients with PCOS expression difference of expression and regulation of mi RNA pathway by experimental verification.【Results】(1) The restropective analysis of clinical cases. Regarding anthropometric and clinical variables and as expected from design, we found no differences in age, estradiol level(E2) PRL level, and FSH level between PCOS patients and controls. However, the body mass index(BMI), serum testosterone(T) concentration, serum glucose(Glu) concentration, serum insulin(INS) concentration, and LH level were significantly higher in PCOS women.(2) The laboratory investigation of mi RNA in GCs. 1)Compared with controls, a total of 59 known mi RNA were identified that differentially expressedin PCOS cumulus granulosa cells, including 21 mi RNAs increase and 38 mi RNAs decrease. 2)Moreover, the novel mi RNAs were predicted in PCOS and control cumulus granulosa cells. 3)The potential regulating roles of mi RNA in pathophysiology of PCOS were analyzed by GO and KEGG pathway annotation, and several important processes were identified to be targeted by the differentially expressed mi RNAs, such as Notch signaling, regulation of hormone, and energy metabolism. 4)Furthermore, Notch3 and MAPK3,the members of Notch signaling and ERK-MAPK pathway, were demonstrated to be regulated by mi R-483-5p were directly targeted by mi R-483-5p.【Conculsion】 Our data suggested that mi RNAs and their targeted pathways(e.g. Notch signaling pathway) play important roles in the etiology and pathophysiology of PCOS, and provides novel candidates for molecular biomarkers or treatment targets in the research of female infertility associated to PCOS.