Analysis Of Aberrant Non-coding RNA And Mechanism In Primary Ovarian Insufficiency

PartⅠMicroRNA-22-3p is down-regulated in the plasma of patients with Primary Ovarian InsufficiencyBackground:Premature ovarian insufficiency （POI）, defined as cessation of menstruation before the expected age of menopause, is one of the most common endocrinopathy for women of child bearing age. The underlying explanation for POI remains to be elucidated in most cases. MicroRNAs （miRNAs） are a class of small （21-24 nucleotides） non-coding RNA molecules that diversely regulate gene expression post-transcriptionally through cleavage or translational repression of specific target messenger RNAs （mRNAs）. Aberrant expression and dysfunction of miRNAs not only relate to tumorigenesis, neurological, cardiovascular, and metabolic disorders, but also contribute to gynecological malignancies, ectopic pregnancy and endometriosis. However, the contribution of miRNAs in the pathogenesis of POI remains elusive.Objective:To determine whether plasma microRNAs （miRNAs） are differentially expressed between women with and without premature ovarian insufficiency （POI）, and to uncover the association of miRNAs with risk of POI.Methods:A total of 140 individuals with POI and 140 age-and body mass index-matched control subjects of Han Chinese ancestry were recruited. Firstly, plasma samples of 10 patients and 10 matched controls were collected and profiled by microarray to identified differentially expressed miRNAs; secondly, quantification of selected miRNAs was replicated by quantitative RT-PCR in additional 50 patients and 50 controls; lastly, independent validation was performed in 80 patients and 80 controls.Results:Fifty-one differentially expressed miRNAs were identified by chip-based discovery stage between 10 patients with POI and 10 controls, among which 9 miRNAs （let-7b-5p, let-7c, miR-15b-5p, miR-22-3p, miR-23a-3p, miR-23b-3p, miR-24-3p, miR-151a-5p and miR-151b） were selected and validated. The relative expression level of miR-22-3p was significantly down-regulated in POI compared with controls. MiR-22-3p yielded a receiver operating characteristic （ROC） curve area of 0.668 （95% confidence interval:0.602-0.733） in discriminating POI from controls. In addition, logistic binary regression analysis and linear regression analysis showed the miR-22-3p was a protective factor for POI （odds ratio:0.766,95%CI:0.643-0.912）, and negatively associated with serum FSH. Furthermore, bioinformatics analysis indicated that the target function of miR-22-3p was involved in apoptosis, endocytosis, and tumorigenesis.Conclusion:Mir-22-3p showed a lower expression level in POI and was modestly effective in distinguishing POI from controls. The decreased expression of miR-22-3p in plasma of POI may reflect the diminished ovarian reserve and be a consequence of the pathological process of POI.Part ⅡThe role and mechanism of microRNA-379-5p in the pathogenesis of biochemical Primary Ovarian InsufficiencyBackground:Throughout the development oocyte, there is an interdependence between the oocyte and its surrounding granulosa cells, the support of which is essential to provide the oocyte with nutrients and growth regulators. Evidence suggests that dysfunction of granulosa cells may contribute to the abnormal folliculogenesis observed in patients with POL MicroRNAs （miRNAs） are small, noncoding RNAs that negatively regulate gene expression post-transcriptionally. Whether differentially expressed miRNAs in granulosa cells contribute to the pathogenesis of POI remains unknown.Objective:To delineate the expression profile of miRNAs in granulosa cells in patients with biochemical POI. To determine whether certain miRNAs are involved in the ovarian dysfunction of biochemical POI and the mechanism of DNA repair deficiency of granulosa cells.Methods:A total of 50 individuals with biochemical POI and 50 age and body mass index matched control subjects of Han Chinese women were recruited. Granulosa cells of 10 patients and 10 matched controls were collected and profiled by microarray to identified differentially expressed miRNAs and mRNAs, and quantification of selected miRNAs and mRNAs was verified by quantitative RT-PCR. KGN granulosa cells were cultured for proliferation assays after overexpression of miR-379-5p with or without UV/H2O2 treatment. Protein expression analysis and luciferase assays were performed to confirm the role of miR-379-5p.Results:MiR-379-5p expression was up-regulated in granulosa cells of biochemical POI patient. Its putative targets, PARP1 and XRCC6 gene, were down-regulated accordingly. Overexpression of miR-379-5p decreased PARP1 and XRCC6 expression at both the mRNA and protein levels. Luciferase reporter assay confirmed PARP1 and XRCC6 acting as direct targets of miR-379-5p. Overexpression of miR-379-5p in KGN cells suppressed cell proliferation and sensitized KGN cells to UV/H2O2 damage, knocking down PARP1 and XRCC6 inhibited cell proliferation in granulosa cells and impaired DNA repair function.Conclusion:These findings indicate that miR-379-5p suppresses cell proliferation and sensitizes granulosa cells to UV/H2O2 damage by targeting the PARP1 and XRCC6 transcript. This might provide a new insight into the dysfunction of granulosa cells in biochemical POI.PartⅢDifferentially expressed Lon-noncoding RNAs in granulosa cells of biochemical POI patientsBackground:LncRNAs are a heterogeneous group of non-coding transcripts more than 200 nt long that are involved in many biological processes. This class of ncRNA makes up the largest portion of the mammalian non-coding transcriptome. Various mechanisms of transcriptional regulation of gene expression by lncRNAs have been proposed. Current results indicate important functions for lncRNAs in organ development, immunity, organismal viability, and in human diseases. However, the contribution of lncRNAs in the pathogenesis of POI remains unclear.Objective:To delineate the lncRNA expression profile in granulosa cells and to determine the deferentially expressed lncRNAs in biochemical POI patients.Methods:A total of 50 individuals with biochemical POI and 50 age-and body mass index-matched control subjects of Han Chinese ancestry were recruited. Granulosa cells of 10 patients and 10 matched controls were collected and profiled by microarray to identified differentially expressed lncRNAs, quantification of selected lncRNAs was replicated by quantitative RT-PCR in additional 32 patients and 32 controls.Results:Six hundred and seventy-seven differentially expressed lncRNAs were identified by chip-based discovery stage between 10 patients with biochemical POI and 10 controls, among which 14 miRNAs were selected and validated. The relative expression level of TCONS₀0010009, TCONS₀0014547, NR₀40662.1, ENST00000520306, ENST00000420313 and ENST00000526225 were significantly down-regulated in biochemical POI compared with controls.Conclusion:There are many of differentially expressed lncRNAs in granulosa cells of biochemical POI patients, indicating the plausible function of lncRNAs in the pathogenesis of POI.