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MiR-140 targets RAP2A to enable the proliferation of insulin-treated ovarian granulosa cells.

ABSTRACT: BACKGROUND:We elucidated the role of specific MicroRNAs (miRNAs) in the development of polycystic ovary syndrome (PCOS) and explained the changes in the proliferation of granulosa cells. Excised ovarian cortex specimens were collected for miRNA profiling analysis (n?=?20 PCOS females and 5 non-PCOS females). Insulin-treated ovarian granulosa cells isolated from mice were used for mechanical studies. RESULTS:High miR-140 expression was observed in PCOS samples and insulin-treated granulosa cells compared to that in non-PCOS and unstimulated cells, respectively. However, the Ras-related protein Rap-2a precursor (RAP2A) was downregulated in in PCOS. MTT assay and EdU staining showed that an miR-140 inhibitor attenuated viability in insulin-treated granulosa cells; cell viability increased with miR-140 overexpression. Reduced expression of miR-140 and the expression of the miR-140 mimic resulted in marked cell apoptosis, as evidenced by the results of PI flow cytometry and Annexin V-FITC; miR-140 overexpression results in downregulated RAP2A expression, and the miR-140 mimic directly bound to the RAP2A 3'-UTR, causing increase in RAP2A levels in insulin-treated granulosa cells; RNA-mediated silencing of RAP2A in insulin-treated granulosa cells restored cell proliferation and apoptosis to normal levels. Phosphorylated AKT was found to be negatively regulated through cross-talk between miR-140 and RAP2A. CONCLUSIONS:In conclusion, PCOS ovarian cortex specimens and insulin-treated granulosa cells showed elevated expression of miR-140, which could lead to increased proliferation and reduced apoptosis of cells by targeting RAP2A. This study may pave the way for future research on the properties of granulosa cells in PCOS.

SUBMITTER: Xiong Z

PROVIDER: S-EPMC7003402 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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MiR-140 targets RAP2A to enable the proliferation of insulin-treated ovarian granulosa cells.

Xiong Zhengfang Z Li Bing B Wang Wenjuan W Zeng Xianghui X Li Binye B Jian Shengyan S Wang Liyun L

Journal of ovarian research 20200205 1

<h4>Background</h4>We elucidated the role of specific MicroRNAs (miRNAs) in the development of polycystic ovary syndrome (PCOS) and explained the changes in the proliferation of granulosa cells. Excised ovarian cortex specimens were collected for miRNA profiling analysis (n = 20 PCOS females and 5 non-PCOS females). Insulin-treated ovarian granulosa cells isolated from mice were used for mechanical studies.<h4>Results</h4>High miR-140 expression was observed in PCOS samples and insulin-treated g ...[more]

PMID: 32024547

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Project description:BackgroundThe expression of miR-140-5p increased in the brain tissue of a bilateral common carotid artery ligation model, while the overexpression of miR-140-5p significantly decreased the number of neurons. The luciferase report experiment in the previous study proved that miR-140-5p negatively regulated one of the potential targets of Prospero-related homeobox 1 (Prox1). Therefore, we want to investigate the effect of miR-140-5p on the proliferation and differentiation of neural stem cells (NSCs) and the underlying mechanism.MethodsPrimary NSCs were extracted from pregnant ICR mice aged 16-18 days and induced to differentiate. After transient transfection with miR-140-5p mimic and inhibitor into NSCs, the cells were divided into five groups: blank, mimic normal control, mimic, inhibitor normal control, and inhibitor. Cell Counting Kit-8 (CCK-8) and 5-Bromo-2-deoxyUridine (BrDU), Ki-67 were used, and the diameter of neural spheres was measured to observe proliferation ability 48 h later. Doublecortin (DCX), glial fibrillary acidic protein (GFAP), microtubule-associated proteins 2 (MAP-2), synapsin I (SYN1), and postsynaptic density protein-95 (PSD-95) were stained to identify the effect of miR-140-5p on the differentiation ability of NSCs into neural precursor cells, astrocytes, and neurons and the expression of synapse-associated proteins. The expression of miR-140-5p, Prox1, p-ERK1/2, and ERK1/2 was analyzed by real time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis.ResultsWhile the expression of miR-140-5p decreased after NSC differentiation (P<0.05), the results of CCK-8, BrDU, and Ki-67 staining showed no significant difference in cell viability and the percentage of NSCs with proliferation ability (P>0.05). However, the neural spheres were shorter in the miR-140-5p overexpression group (P<0.05) and the expression of DCX, MAP2, synapsin I, and PSD-95 decreased, while the expression of GFAP increased after differentiation in the mimic group (P<0.05). In addition, the expression of Prox1 decreased and the expression of p-ERK1/2 protein increased (P<0.05), but the expression of ERK1/2 showed no significant difference (P>0.05) in the miR-140-5p overexpression group.ConclusionsMiR-140-5p reduced the proliferation rate of NSCs, inhibited their differentiation into neurons, produced synapse-associated proteins, and promoted their differentiation into astrocytes. MiR-140-5p negatively regulated downstream target Prox1 and activated the ERK/MAPK signaling pathway.

Project description:BackgroundIn mammals, it is known that the estradiol-17β (E2) is mainly synthetized in ovarian granulosa cells (GCs), and the excessive apoptosis of GCs induces the follicular atresia. Many studies have implicated the essential role of KISS1, with the pro-synthetic effect of E2 and the anti-apoptotic effect on GCs, in the mammalian folliculogenesis, and several STAT4 potential binding sites were previously predicted on the promoter of KISS1 in pigs. However, the biological effects of STAT4 on GCs and the molecular regulation between STAT4 and KISS1 remained largely unknown.MethodsUsing the porcine GCs as the cellular model, the overexpression plasmid, small interfering RNA, 5'-deletion and luciferase assay were applied to investigate the molecular mechanisms for STAT4 regulating the expression of KISS1.ResultsIn this study, the STAT4 negatively regulated the mRNA and protein levels of KISS1 in porcine GCs, and the mRNA level of STAT4 was observed to significantly decrease from immature to mature follicles, which was inversed with that of KISS1. The relative luciferase activity of KISS1 promoter was significantly increased with deletion of the fourth potential binding site (- 305/- 295), and ChIP further confirmed that the STAT4 bound at - 305/- 295 region of KISS1. Besides, the STAT4 significantly regulated the mRNA levels of PDK1, FOXO3 and TSC2 of PI3K signaling pathway to promote the cell apoptosis and the percentage of cells at G0/G1 phase of cell cycle in GCs. Alternatively, the STAT4 significantly decreased the mRNA levels of CYP17, 3B-HSD, 17B-33 HSD, ESR1, and ESR2, as well as the concentration of E2 in GCs. Furthermore, interfering with the expression of STAT4 was observed to significantly stimulate the pro-synthetic effect of E2 and anti-apoptotic effect of KISS1 in GCs.ConclusionsCollectively, the STAT4 might directly target at - 305/- 295 region of KISS1 to negatively regulate the transcription of KISS1, promote the cell apoptosis via PI3K signaling pathway, suppress the synthesis of E2 through the estrogen signaling pathway in porcine GCs. These proposed works could provide useful insight in further investigations on the molecular functionalities of STAT4 and KISS1 in the folliculogenesis of mammals.

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MiR-140 targets RAP2A to enable the proliferation of insulin-treated ovarian granulosa cells.

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MiR-140 targets RAP2A to enable the proliferation of insulin-treated ovarian granulosa cells.

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