| BackgroundOvarian reserve reflects the growth and development of follicles and their ability to produce mature oocytes in the ovarian cortex.It is determined by the quantity and quality of follicles stored in the ovary and can represent the reproductive potential of women.Diminished ovarian reserve(DOR)refers to the decrease in the number of recruited follicles and the quality of oocytes in the ovaries of women of childbearing age.It is a transitional disease between normal ovarian function and premature ovarian failure.Devine et al.Found that the prevalence of DOR increased from 19%to 26%among patients treated with assisted reproductive technology in the United States from 2004 to 2011.At present,the etiology of DOR is still not completely clear,but genetic factors,environmental factors,ovarian injury caused by ovarian surgery and radiotherapy and chemotherapy,abnormal expression of TGF can cause abnormal follicular development in the ovary,excessive depletion of the primordial follicle bank,inhibited the development of primary and secondary follicles,accelerated follicular atresia,and eventually led to the occurrence of DOR.The signal transduction and interaction between granulosa cells and oocytes regulate follicular development,and the proliferation,apoptosis and hormone secretion of granulosa cells also directly affect the whole process of follicular development.Therefore,the study of granulosa cell function and follicular development is helpful to reveal the molecular mechanism of DOR.microRNA(miRNA)is a kind of endogenous single-stranded small RNA with 22～25 nucleotides(BP).The genes expressing miRNAs are located in the non-coding region of chromosome,which is conservative in the process of biological evolution.miRNAs can effectively regulate gene expression through posttranscriptional negative regulation,participate in intracellular signal transduction,affect cell proliferation,differentiation,apoptosis and other physiological activities,as well as tumorigenesis,autoimmune and other pathological processes.In the field of reproductive medicine,the abnormal expression of miRNAs can affect all aspects of follicular development,such as primordial follicle recruitment,granulosa cell proliferation and differentiation,steroid hormone synthesis and secretion,oocyte maturation,ovulation and luteinization.The direct cause of DOR is follicular dysplasia,so miRNAs play an important role in the pathogenesis of DOR.Our group’s previous pilot study showed that the expression of miR-338-3p in granulosa cells of patients with premature ovarian insufficiency(POI)was higher than that of patients with normal ovarian function.Because of the above results,this study took human primary granulosa cells and KGN cells as the research objects to explore the relationship between the regulation of miR-338-3p on the function of granulosa cells and the decline of ovarian function,and took mice as the research objects to explore the effect of miR-338-3p on follicular development.Objective1.To explore the expression and localization of miR-338-3p in granulosa cells and mouse ovaries,and to verify the differential expression of miR-338-3p in granulosa cells of patients with ovarian dysfunction and normal ovarian function;2.To explore the regulation of miR-338-3p on the proliferation and secretion of granulosa cells;3.To explore the effect of miR-338-3p on early follicular development in mice;4.To explore the mechanism of miR-338-3p involved in the proliferation of granulosa cells mediated by TGF-β.MethodGranulosa cells were collected from patients with ovarian dysfunction and normal ovarian function who were treated with IVF in our hospital,RT-qPCR was used to detect the expression of miR-338-3p;fluorescence in situ hybridization was used to identify the expression of miR-338-3p in human granulosa cells and mouse ovary;CCK-8(cell counting kit-8)kit was used to detect the regulation of miR-338-3p on proliferation and apoptosis of KGN cells;immunofluorescence was used to detect the expression of Ki-67 in primary granulosa cells after overexpression and low expression of miR-338-3p;Western blot was used to detect the expression of Bcl-2,p53 and aromatase in primary granulosa cells.miR-338-3p-adenovirus was used to construct the overexpression culture system in vitro.The development of mouse follicles was observed by HE staining.The primary granulosa cells were treated with different concentrations of TGF-β(0ng/ml,1ng/ml,2ng/ml),and the expression of miR-338-3p was detected by RT-qPCR.The effect of miR-338-3p on the proliferation of granulosa cells was detected by CCK-8 kit.Result1.miR-338-3p was located in the cytoplasm of human granulosa cells,mouse granulosa cells and oocytes.The expression level of miR-338-3p in granulosa cells of DOR patients was significantly higher than that in the control group with normal ovarian function;2.After miR-338-3p overexpression treatment,the proliferation of kgn cells decreased significantly at 48h and 72h(P<0.05),while the proliferation of kgn cells increased significantly at 24h and 72h after miR-338-3p down-regulation(P<0.05);3.When the cell density was basically the same,the expression of Ki-67 in the nucleus of miR-338-3p overexpression group was significantly lower than that of the control group,while the expression of Ki-67 was significantly increased after miR-338-3p down-regulation;4.After up regulating the expression of miR-338-3p,the level of BCL2 protein decreased and the level of p53 protein increased(P<0.05);after down regulating the expression of miR-338-3p,the level of BCL2 protein increased and the level of p53 protein decreased(P<0.05);5.miR-338-3p can down regulate the expression level of aromatase protein and inhibit the secretion of estradiol in primary granulosa cells.Down regulation of miR-338-3p can increase the secretion level of estradiol;6.After overexpression of miR-338-3p-adenovirus,the primordial follicle development of D1 mice stagnated,while the follicles of the blank control group continued to develop into primary follicles;after overexpression of miR-338-3p-adenovirus,the ovaries of D4 mice still stagnated in the primary follicle stage compared with the fresh control group,while the follicles of blank control group continued to develop,with more secondary follicles;7.The expression of miR-338-3p in granulosa cells gradually decreased with the increase of TGF-β concentration;TGF-β could promote the proliferation of KGN cells.After up regulating the expression of mir-338-3p in KGN cells,the proliferation ability of kgn cells was decreased compared with the non-up regulating group,but it was still enhanced compared with the up regulating group.ConclusionThe expression of miR-338-3p in granulosa cells from patients with ovarian dysfunction was significantly increased.Meanwhile,the increase of miR-338-3p could inhibit the proliferation and hormone secretion of granulosa cells,and slow down the early follicular development of mice.TGF-β can inhibit the expression of miR-338-3p in primary granulosa cells,and promote granulosa cell proliferation and follicle maturation by down regulating the expression of miR-3 3 8-3p in granulosa cells. |
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