LncRNA FDNCR promotes apoptosis of granulosa cells by targeting the miR-543-3p/DCN/TGF-β signaling pathway in Hu sheep.
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ABSTRACT:
SUBMITTER: Yao X
PROVIDER: S-EPMC7973142 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAtresia and degeneration, a follicular developmental fate that reduces female fertility and is triggered by granulosa cell (GC) apoptosis, have been induced by dozens of miRNAs. Here, we report a miRNA, miR-423, that inhibits the initiation of follicular atresia (FA), and early apoptosis of GCs.ResultsWe showed that miR-423 was down-regulated during sow FA, and its levels in follicles were negatively correlated with the GC density and the P4/E2 ratio in the follicular fluid in vivo. The in vitro gain-of-function experiments revealed that miR-423 suppresses cell apoptosis, especially early apoptosis in GCs. Mechanically speaking, the miR-423 targets and interacts with the 3'-UTR of the porcine SMAD7 gene, which encodes an apoptosis-inducing factor in GCs, and represses its expression and pro-apoptotic function. Interestingly, FA and the GC apoptosis-related lncRNA NORHA was demonstrated as a ceRNA of miR-423. Additionally, we showed that a single base deletion/insertion in the miR-423 promoter is significantly associated with the number of stillbirths (NSB) trait of sows.ConclusionThese results demonstrate that miR-423 is a small molecule for inhibiting FA initiation and GC early apoptosis, suggesting that treating with miR-423 may be a novel approach for inhibiting FA initiation and improving female fertility.
| S-EPMC10696705 | biostudies-literature
Project description:BackgroundThe intricate interplay of gene expression within ovarian granulosa cells (GCs) is not fully understood. This study aimed to investigate the miRNA regulatory mechanisms of ferroptosis during the process of follicle development in lamb GCs.MethodsEmploying transcriptome sequencing, we compared differentially expressed mRNAs (DE-mRNAs) and miRNAs (DE-miRNAs) in GCs from lambs treated with follicle-stimulating hormone (FL) to untreated controls (CL). We further screened differentially expressed ferroptosis-related genes and identified potential miRNA regulatory factors. The expression patterns of HMOX1 and miRNAs in GCs were validated using qRT‒PCR and Western blotting. Additionally, we investigated the regulatory effect of oar-miR-134-3p on HMOX1 and its function in ferroptosis through cell transfection and erastin treatment.ResultsWe identified a total of 4,184 DE-mRNAs and 304 DE-miRNAs. The DE-mRNAs were mainly enriched in ferroptosis, insulin resistance, and the cell cycle. Specifically, we focused on the differential expression of ferroptosis-related genes. Notably, the ferroptosis-related genes HMOX1 and SLC3A2, modulated by DE-miRNAs, were markedly suppressed in FLs. Experimental validation revealed that HMOX1 was significantly downregulated in FL and large follicles, while oar-miR-134-3p was significantly upregulated compared to that in the CLs. HMOX1 expression was regulated by the targeting effect of oar-miR-134-3p. Functional assays further revealed that modulation of oar-miR-134-3p influenced HMOX1 expression and altered cellular responses to ferroptosis induction by erastin.ConclusionThis study suggested that oar-miR-134-3p and HMOX1 may be one of the pathways regulating ferroptosis in GCs. This finding provides new clues to understanding the development and regulatory process of follicles.
| S-EPMC10763389 | biostudies-literature
Project description:Ischemic cardiac disease is the leading cause of morbidity and mortality in the world. Despite the great efforts and progress in cardiac research, the current treatment of cardiac ischemia reperfusion injury (I/RI) is still far from being satisfactory. This study was performed to investigate the role of long non-coding RNA (lncRNA) H19 in regulating myocardial I/RI. We found that H19 expression was downregulated in the I/R hearts of mice and cardiomyocytes treated with H2O2. Overexpression of H19 alleviated myocardial I/RI of mice and cardiomyocyte injury induced by H2O2. We found that H19 functioned as a competing endogenous RNA of miR-877-3p, which decreased the expression of miR-877-3p through the base-pairing mechanism. In parallel, miR-877-3p was upregulated in H2O2-treated cardiomyocytes and mouse ischemia reperfusion (I/R) hearts. miR-877-3p exacerbated myocardial I/RI and cardiomyocyte apoptosis. We further established Bcl-2 as a downstream target of miR-877-3p. miR-877-3p inhibited the mRNA and protein expression of Bcl-2. Furthermore, H19 decreased the Bcl-2/Bax ratio at mRNA and protein levels, cytochrome c release, and activation of caspase-9 and caspase-3 in myocardial I/RI mice, which were canceled by miR-877-3p. In summary, the H19/miR-877-3p/Bcl-2 pathway is involved in regulation of mitochondrial apoptosis during myocardial I/RI, which provided new insight into molecular mechanisms underlying regulation of myocardial I/RI.
| S-EPMC6612907 | biostudies-other
Project description:Abnormal sexual maturity exhibits significant detrimental effects on adult health outcomes, and previous studies have indicated that targeting histone acetylation might serve as a potential therapeutic approach to regulate sexual maturity. However, the mechanisms that account for it remain to be further elucidated. Using the mouse model, we showed that Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, downregulated the protein level of Hdac1 in ovaries to promote the apoptosis of granulosa cells (GCs), and thus arrested follicular development and delayed sexual maturity. Using porcine GCs as a cell model, a novel sexual maturity-associated lncRNA, which was named as the stimulatory factor of follicular development (SFFD), transcribed from mitochondrion and mediated by HDAC1, was identified using RNA sequencing. Mechanistically, HDAC1 knockdown significantly reduced the H3K27ac level at the -953/-661 region of SFFD to epigenetically inhibit its transcription. SFFD knockdown released miR-202-3p to reduce the expression of cyclooxygenase 1 (COX1), an essential rate-limited enzyme involved in prostaglandin synthesis. This reduction inhibited the proliferation and secretion of 17β-estradiol (E2) while promoting the apoptosis of GCs. Consequently, follicular development was arrested and sexual maturity was delayed. Taken together, HDAC1 knockdown-mediated SFFD downregulation promoted the apoptosis of GCs through the miR-202-3p-COX1 axis and lead to delayed sexual maturity. Our findings reveal a novel regulatory network modulated by HDAC1, and HDAC1-mediated SFFD may be a promising new therapeutic target to treat delayed sexual maturity.
| S-EPMC10706290 | biostudies-literature
Project description:Background:Breast cancer is the most common female malignancy with high invasion and metastasis abilities. Studies have shown that long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 gene (PVT1) is an oncogene and is positively correlated with progression and metastasis of breast tumors. However, the detailed mechanism of PVT1 in breast cancer tumorigenesis is not fully understood. Methods:Real-time polymerase quantitative chain reaction (RT-qPCR) was performed to identify the expression levels of PVT1, miR-543 and trichorhinophalangeal syndrome-1 gene (TRPS1) in breast cancer tissues and cells. Cell proliferation was measured by plate clone formation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay. Apoptosis and motility of MCF-7 and MDA-MB-436 cells were assessed with flow cytometry assay and transwell migration and invasion analyses, respectively. In addition, a model was established to probe the function of PVT1 silencing in vivo. The target relationship among PVT1, miR-543 or TRPS1 was confirmed by dual-luciferase reporter analysis, RNA immunoprecipitation (RIP) and RNA pull down assays. The protein expression level of TRPS1 was evaluated with Western blot assay. Results:PVT1 expression was upregulated in breast cancer tissues and cell lines. In addition, PVT1 silencing inhibited breast cancer cell growth and motility, while increased apoptosis. Meanwhile, the effects of PVT1 or miR-543 could be reversed by introducing overexpressed plasmid of miR-543 or TRPS1 in breast cancer cell lines, respectively. Conclusion:Knockdown of PVT1 repressed breast cancer cell growth and motility, and induced apoptosis in vitro and reduced tumor volume and weight in vivo. Mechanically, the overexpression of PVT1 enhanced TRPS1 level by negatively targeted miR-543 in breast cancer.
| S-EPMC7493016 | biostudies-literature
Project description:BackgroundIn our previous investigation, we revealed a significant increase in the expression of microRNA-6881-3p (miR-6881-3p) in follicular fluid granulosa cells (GCs) from women with diminished ovarian reserve (DOR) compared to those with normal ovarian reserve (NOR). However, the role of miR-6881-3p in the development of DOR remains poorly understood.ObjectiveThis study aimed to elucidate the involvement of miR-6881-3p in the regulation of granulosa cells (GCs) function and the pathogenesis of DOR.Materials and methodsInitially, we assessed the expression levels of miR-6881-3p in GCs obtained from human follicular fluid in both NOR and DOR cases and explored the correlation between miR-6881-3p expression and clinical outcomes in assisted reproduction technology (ART). Bioinformatic predictions and dual-luciferase reporter assays were employed to identify the target gene of miR-6881-3p. Manipulation of miR-6881-3p expression was achieved through the transfection of KGN cells with miR-6881-3p mimics, inhibitor, and miRNA negative control (NC). Following transfection, we assessed granulosa cell apoptosis and cell cycle progression via flow cytometry and quantified target gene expression through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Finally, we examined the correlation between target gene expression levels in GCs from NOR and DOR patients and their association with ART outcomes.ResultsOur findings revealed elevated miR-6881-3p levels in GCs from DOR patients, which negatively correlated with ovarian reserve function and ART outcomes. We identified a direct binding interaction between miR-6881-3p and the 3'-untranslated region of the SMAD4. Transfection with miR-6881-3p mimics induced apoptosis in KGN cell. Furthermore, miR-6881-3p expression negatively correlated with both mRNA and protein levels of the SMAD4. The mRNA and protein levels of SMAD4 were notably reduced in GCs from DOR patients, and SMAD4 mRNA expression positively correlated with ART outcomes. In addition, the mRNA levels of FSHR, CYP11A1 were notably reduced after transfection with miR-6881-3p mimics in KGN cell, while LHCGR notably increased. The mRNA and protein levels of FSHR, CYP11A1 were notably reduced in GCs from DOR patients, while LHCGR notably increased.ConclusionThis study underscores the role of miR-6881-3p in directly targeting SMAD4 mRNA, subsequently diminishing granulosa cell viability and promoting apoptosis, and may affect steroid hormone regulation and gonadotropin signal reception in GCs. These findings contribute to our understanding of the pathogenesis of DOR.
| S-EPMC10832098 | biostudies-literature
Project description:The aim of this study was to investigating whether lncRNA H19 promotes myocardial fibrosis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis. Patients with atrial fibrillation (AF) and healthy volunteers were included in the study, and their biochemical parameters were collected. In addition, pcDNA3.1-H19, si-H19, and miR-29a/b-3p mimic/inhibitor were transfected into cardiac fibroblasts (CFs), and proliferation of CFs was detected by MTT assay. Expression of H19 and miR-29a/b-3p were detected using real-time quantitative polymerase chain reaction, and expression of α-smooth muscle actin (α-SMA), collagen I, collagen II, matrix metalloproteinase-2 (MMP-2), and elastin were measured by western blot analysis. The dual luciferase reporter gene assay was carried out to detect the sponging relationship between H19 and miR-29a/b-3p in CFs. Compared with healthy volunteers, the level of plasma H19 was significantly elevated in patients with AF, while miR-29a-3p and miR-29b-3p were markedly depressed (P < 0.05). Serum expression of lncRNA H19 was negatively correlated with the expression of miR-29a-3p and miR-29b-3p among patients with AF (rs = -0.337, rs = -0.236). Moreover, up-regulation of H19 expression and down-regulation of miR-29a/b-3p expression facilitated proliferation and synthesis of extracellular matrix (ECM)-related proteins. SB431542 and si-VEGFA are able to reverse the promotion of miR-29a/b-3p on proliferation of CFs and ECM-related protein synthesis. The findings of the present study suggest that H19 promoted CF proliferation and collagen synthesis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis, and provide support for a potential new direction for the treatment of AF.
| S-EPMC8926865 | biostudies-literature
Project description:ObjectiveThis study explores LncRNA TM1-3P effects on the proliferation, apoptosis, and inflammatory response of fibroblasts in osteoarthritis (OA) and its underlying mechanism.MethodsBioinformatics was performed to analyze OA disease-related genes, miRNA profiles, and function. The targeted regulation of LncRNA TM1-3P and miR-144-3p, ONECUT2 and miR-144-3p were analyzed by dual luciferase reporter gene assay, RNA Binding Protein Immunoprecipitation (RIP), and RNA pull down. Histopathological morphology of the knee joint was observed by hematoxylin-eosin (HE) and Annona Red O/Fast Green. The expressions of mRNAs and proteins were detected by RT-qPCR, Western blot, and immunohistochemistry. Unpaired T test was used between groups, and the one-way analysis of variance of repeated measurement data was applied for multi-group comparison, following Tukey's post-test.ResultsONECUT2 and Smurf2 genes were significantly elevated in the osteoarthritis group compared with the normal group (P < 0.001, P < 0.001). Expressions of ONECUT2 and LncRNA TM1-3P were increased, and expression of miR-144-3p was decreased in interleukin (IL)-1β-induced human fibroblast synovial cells (hFSCs) (mRNA: 1.06 ± 0.24 vs. 3.29 ± 0.73, proteins: 0.22 ± 0.03 vs. 0.46 ± 0.22, 1.23 ± 0.22 vs. 3.76 ± 0.73, 1.06 ± 0.25 vs. 0.37 ± 0.13, P < 0.01, P < 0.001, P < 0.01, P < 0.05). Overexpression of miR-144-3p down-regulated the ONECUT2 expression, reduced cell proliferation, promoted apoptosis in hFSCs induced by IL-1β (mRNA: 0.89 ± 0.14 vs. 0.15 ± 0.01, P < 0.05; proteins: 0.46 ± 0.01 vs. 0.23 ± 0.01, P < 0.001; CCK8: 1.88 ± 0.07 vs. 1.65 ± 0.07; P < 0.05; EDU: 55.82 ± 1.44 vs 40.57 ± 2.24, P < 0.05; apoptosis: 10.57 ± 0.79 vs 16.36 ± 0.35, P < 0.0001). Overexpression of LncRNA TM1-3P up-regulated the expression of ONECUT2, promoted cell proliferation, and inhibited apoptosis (mRNA: 0.9 ± 0.09 vs 1.94 ± 0.12, P < 0.05; proteins: 0.61 ± 0.05 vs 0.76 ± 0.03, P > 0.05; CCK8: 2.07 ± 0.05 vs 2.47 ± 0.06; P < 0.01; EDU: 52.67 ± 1.17 vs 60.06 ± 3.24, P < 0.05; apoptosis: 10.57 ± 0.79 vs 16.36 ± 0.35, P < 0.001), which were reversed by the overexpression of miR-144-3p treatment (mRNA: 1.82 ± 0.07 vs 0.31 ± 0.07, P < 0.0001; proteins: 0.74 ± 0.02 vs 0.35 ± 0.01, P < 0.01; CCK8: 2.41 ± 0.01 vs 1.67 ± 0.02; P < 0.0001; EDU: 66.85 ± 2.86 vs 44.68 ± 1.97, P < 0.0001; apoptosis: 7.19 ± 0.19 vs 13.36 ± 0.53, P < 0.0001). Silencing LncRNA TM1-3P attenuated the injury of knee joint tissue, down-regulated the expression of ONECUT2, Smurf2, IL-1β, IL-6, TNF-α, and improved the expression of Rap1 in rats (0.71 ± 0.04 vs 0.48 ± 0.02, 0.68 ± 0.06 vs 0.36 ± 0.02, 0.74 ± 0.03 vs 0.49 ± 0.04, 0.78 ± 0.01 vs 0.54 ± 0.03, 0.68 ± 0.02 vs 0.4 ± 0.04, 0.24 ± 0.01 vs 0.4 ± 0.03, P < 0.05, P < 0.05, P < 0.05, P < 0.01, P < 0.01, P < 0.05).ConclusionLncRNA TM1-3P improved inflammation and damage of knee joints in OA rats through miR-144-3p/ONECUT2 axis, providing a new theoretical basis for gene therapy of OA.
| S-EPMC9627084 | biostudies-literature
Project description:The proliferation and apoptosis of granulosa cells (GCs) affect follicle development and reproductive disorders, with microRNAs playing a crucial regulatory role. Previous studies have shown the differential expression of miR-128-3p at different stages of goat follicle development, which suggests its potential regulatory role in follicle development. In this study, through the Cell Counting Kit-8 assay, the EDU assay, flow cytometry, quantitative real-time polymerase chain reaction, Western blot, and the dual-luciferase reporter assay, we used immortal human ovarian granulosa tumor cell line (KGN) cells as materials to investigate the effects of miR-128-3p and its predicted target gene growth hormone secretagogue receptor (GHSR) on GC proliferation and apoptosis. The results show that overexpression of miR-128-3p inhibited the proliferation of KGN cells, promoted cell apoptosis, and suppressed the expression of proliferating cell nuclear antigen (PCNA) and B-cell lymphoma-2 (BCL2) while promoting that of Bcl-2 associated X protein (BAX). The dual-luciferase reporter assay revealed that miR-128-3p bound to the 3' untranslated region sequence of GHSR, which resulted in the inhibited expression of GHSR protein. Investigation of the effects of GHSR on GC proliferation and apoptosis revealed that GHSR overexpression promoted the expression of PCNA and BCL2, enhanced GC proliferation, and inhibited cell apoptosis, whereas the opposite effects were observed when GHSR expression was inhibited. In addition, miR-128-3p and GHSR can influence the expression of extracellular signal-regulated kinase 1/2 protein. In conclusion, miR-128-3p inhibits KGN cell proliferation and promotes cell apoptosis by downregulating the expression of the GHSR gene.
| S-EPMC10931543 | biostudies-literature
Project description:Follicle-stimulating hormone receptor (FSHR) and its intracellular signaling control mammalian follicular development and female infertility. Our previous study showed that FSHR is downregulated during follicular atresia of porcine ovaries. However, its role and regulation in follicular atresia remain unclear. Here, we showed that FSHR knockdown induced porcine granulosa cell (pGC) apoptosis and follicular atresia, and attenuated the levels of intracellular signaling molecules such as PKA, AKT and p-AKT. FSHR was identified as a target of miR-143, a microRNA that was upregulated during porcine follicular atresia. miR-143 enhanced pGC apoptosis by targeting FSHR, and reduced the levels of intracellular signaling molecules. SMAD4, the final molecule in transforming growth factor (TGF)-β signaling, bound to the promoter and induced significant downregulation of miR-143 in vitro and in vivo. Activated TGF-β signaling rescued miR-143-reduced FSHR and intracellular signaling molecules, and miR-143-induced pGC apoptosis. Overall, our findings offer evidence to explain how TGF-β signaling influences and FSHR signaling for regulation of pGC apoptosis and follicular atresia by a specific microRNA, miR-143.
| S-EPMC5260897 | biostudies-literature