Age-associated up-regulation of EGR1 promotes granulosa cell apoptosis during follicle atresia in mice through the NF-κB pathway

Follicular atresia is the main process responsible for the loss of follicles and oocytes from the ovary, and it is the root cause of ovarian aging. Apoptosis of granulosa cells (GCs) is the cellular mechanism responsible for follicular atresia in mammals. Recent advances have highlighted fundamental roles for EGR1 in age-related diseases via the induction of apoptosis. In the present study, we found that the expression of EGR1 was significantly increased in aged mouse ovaries compared with young ovaries. Immunohistochemical analysis revealed strongly positive EGR1 staining in atretic follicles, especially in apoptotic granulosa cells. We further showed that EGR1 up-regulation in mouse primary granulosa cells inhibited cell proliferation and promoted apoptosis. In addition, the promotion of apoptosis in GCs by EGR1 increases over time and with reactive oxygen species (ROS) stimulation. Our mechanistic study suggested that EGR1 regulates GC apoptosis in a mitochondria-dependent manner and that this mainly occurs through the NF-κB signaling pathway. In conclusion, our results suggested that age-related up-regulation of EGR1 promotes GC apoptosis in follicle atresia during ovarian aging.     

Transactivation of MicroRNA-320 by MicroRNA-383 Regulates Granulosa Cell Functions by Targeting E2F1 and SF-1 Proteins

Our previous studies have shown that microRNA-320 (miR-320) is one of the most down-regulated microRNAs (miRNA) in mouse ovarian granulosa cells (GCs) after TGF-β1 treatment. However, the underlying mechanisms of miR-320 involved in GC function during follicular development remain unknown. In this study, we found that pregnant mare serum gonadotropin treatment resulted in the suppression of miR-320 expression in a time-dependent manner. miR-320 was mainly expressed in GCs and oocytes of mouse ovarian follicles in follicular development. Overexpression of miR-320 inhibited estradiol synthesis and proliferation of GCs through targeting E2F1 and SF-1. E2F1/SF-1 mediated miR-320-induced suppression of GC proliferation and of GC steroidogenesis. FSH down-regulated the expression of miR-320 and regulated the function of miR-320 in mouse GCs. miR-383 promoted the expression of miR-320 and enhanced miR-320-mediated suppression of GC proliferation. Injection of miR-320 into the ovaries of mice partially promoted the production of testosterone and progesterone but inhibited estradiol release in vivo. Moreover, the expression of miR-320 and miR-383 was up-regulated in the follicular fluid of polycystic ovarian syndrome patients, although the expression of E2F1 and SF-1 was down-regulated in GCs. These data demonstrated that miR-320 regulates the proliferation and steroid production by targeting E2F1 and SF-1 in the follicular development. Understanding the regulation of miRNA biogenesis and function in the follicular development will potentiate the usefulness of miRNA in the treatment of reproduction and some steroid-related disorders.     

miR-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary

More than 99% of ovarian follicles undergo atresia in mammals, but the mechanism of follicular atresia remains to be elucidated. In this study, we explored microRNA (miRNA) regulation of follicular atresia in porcine ovary. A miRNA expression profile was constructed for healthy, early atretic, and progressively atretic follicles, and the differentially expressed miRNAs were selected and analyzed. We found that miR-26b, which was upregulated during follicular atresia, increased the number of DNA breaks and promoted granulosa cell apoptosis by targeting the ataxia telangiectasia mutated gene directly in vitro.     

MicroRNA-205 affects mouse granulosa cell apoptosis and estradiol synthesis by targeting CREB1

MicroRNA-205 (miR-205) is involved in various physiological and pathological processes, but its biological function in follicular atresia remains unclear. In this study, we investigated miR-205 expression in mouse granulosa cells (mGCs) and analyzed its functions in primary mGCs by performing a series of in vitro experiments. Quantitative real-time polymerase chain reaction showed that miR-205 expression was significantly higher in early atretic follicles and progressively atretic follicles than in healthy follicles. miR-205 overexpression in mGCs significantly promoted apoptosis and caspase-3/9 activities, as well as inhibited estrogen (E2) release and cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1, a key gene in E2 production) expression. Bioinformatics and luciferase reporter assays revealed that the gene encoding cyclic AMP response element (CRE)-binding protein 1 (CREB1) was a direct target of miR-205 in mGCs. CREB1 upregulation partially rescued the effects of miR-205 on apoptosis, caspase-3/9 activities, E2 production, and CYP19A1 expression on mGCs. These results indicate that miR-205 might play an important role in ovarian follicular development and provide new insights into follicular atresia     

PCOS follicular fluid derived exosomal miR-424-5p induces granulosa cells senescence by targeting CDCA4 expression

Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive disease, characterized by increased ovarian androgen biosynthesis, chronic anovulation and polycystic ovaries. The objective of this study was to identify the altered miRNA expression profiles in follicular fluid derived exosomes isolated from PCOS patients and to investigate the molecular functions of exosomal miR-424-5p. Herein, small RNA sequencing showed that twenty-five miRNAs were differentially expressed between control and PCOS group. The alterations in the miRNA profile were related to the endocrine resistance, cell growth and proliferation, cellular senescence and insulin signaling pathway. Among these differentially expressed miRNAs, we found that the expression of miR-424-5p was significantly decreased in PCOS exosomes and primary granulosa cells (GCs). Exosome-enriched miR-424-5p significantly promoted GCs senescence and suppressed cell proliferation. Similar to the results obtained in the cells transfected with miR-424-5p mimic, miR-424-5p mimic significantly decreased cell proliferation ability and induced senescence, but treatment with miR-424-5p inhibitor got the opposite results. In addition, cell division cycle associated 4 (CDCA4) gene displayed an inverse expression pattern to those of miR-424-5p, was identified as the direct target of miR-424-5p. Overexpression of CDCA4 reversed the effects of exosomal miR-424-5p on GCs via activation of Rb/E2F1 signaling pathway. These results demonstrate that exosomal miR-424-5p inhibits GCs proliferation and induces cellular senescence in PCOS through blocking CDCA4-mediated Rb/E2F1 signaling. Our findings provide new information on abnormal follicular development in PCOS.     

Prolactin in follicular fluid and intracellular store calcium in follicular cells are related to morphological signs of ovarian follicle atresia in cows: work in progress

It is known that prolactin (PRL) is the third pituitary hormone serving gonadotropic function in mammals. However, its role in the regulation of ovarian folliculogenesis and, in particular, its relationship to follicular atresia as well as the mechanism of its influence on follicular cells are poorly understood. We investigated PRL levels in follicular fluids (FFs) and intracellular store calcium ([Ca2+]is) in cell walls of bovine ovarian follicles with diameters of 10 to 20 mm and their relationship to follicular atresia. Ovarian follicles were categorized on the basis of macroscopic criteria and of microscopic examination of granulosa cell (GC) smears. Prolactin concentrations in FFs were measured by RIA and levels of [Ca2+]is in follicular cells were determined by using the fluorophore chlortetracycline. Compared to atretic follicles, morphologically normal follicles were characterized by higher concentrations of PRL in FFs (P < 0.001) and lower contents of [Ca2+]is in follicular cells (P < 0.01). Furthermore, follicles containing no more than 20% of pycnotic GCs had higher levels of PRL in their fluids than those containing over 40% of pycnotic GCs (P < 0.05). Finally, the direct effect of PRL on [Ca2+]is content in follicular cells was studied in vitro. Compared to control, PRL decreased (P < 0.001) the levels of [Ca2+]is in the cells after 24 h culture of follicular walls from morphologically normal follicles in TCM 199 supplemented by 10% fetal calf serum. Our findings suggest that the decline of PRL concentrations in FFs and the rise of [Ca2+]is contents in follicular cells are related to atresia of large bovine follicles and that there appears to be a relationship between the two biochemical parameters.     

MiR-92a inhibits porcine ovarian granulosa cell apoptosis by targeting Smad7 gene

Smad7 has a key role in apoptosis of mammalian ovarian granulosa cells (GCs), as it antagonizes and fine-tunes transforming growth factor β (TGFβ) signaling. This study demonstrates that miR-92a regulates GC apoptosis in pig ovaries by targeting Smad7 directly. The expression level of miR-92a was down-regulated in atretic porcine follicles, whereas miR-92a expression led to inhibition of GC apoptosis. The Smad7 gene was identified as a direct target of miR-92a using a dual-luciferase reporter assay. Transfection of GCs with miR-92a mimics decreased Smad7 mRNA and protein levels, whereas expression of an miR-92a inhibitor in GCs had the opposite effect. In addition, knockdown of Smad7 prevented GC apoptosis in cells that expressed the miR-92a inhibitor.     

miRNA profiling of granulosa cell-derived exosomes reveals their role in promoting follicle development

To explore whether granulosa cell (GC)-derived exosomes (GC-Exos) and follicular fluid-derived exosomes (FF-Exos) have functional similarities in follicle development and to establish relevant experiments to validate whether GC-Exos could serve as a potential substitute for follicular fluid-derived exosomes to improve folliculogenesis. GC-Exos were characterized. MicroRNA (miRNA) profiles of exosomes from human GCs and follicular fluid were analyzed in depth. The signature was associated with folliculogenesis, such as phosphatidylinositol 3 kinases-protein kinase B signal pathway, mammalian target of rapamycin signal pathway, mitogen-activated protein kinase signal pathway, Wnt signal pathway, and cyclic adenosine monophosphate signal pathway. A total of five prominent miRNAs were found to regulate the above five signaling pathways. These miRNAs include miRNA-486-5p, miRNA-10b-5p, miRNA-100-5p, miRNA-99a-5p, and miRNA-21-5p. The exosomes from GCs and follicular fluid were investigated to explore the effect on folliculogenesis by injecting exosomes into older mice. The proportion of follicles at each stage is counted to help us understand folliculogenesis. Exosomes derived from GCs were isolated successfully. miRNA profiles demonstrated a remarkable overlap between the miRNA profiles of FF-Exos and GC-Exos. The shared miRNA signature exhibited a positive influence on follicle development and activation. Furthermore, exosomes derived from GCs and follicular fluid promoted folliculogenesis in older female mice. Exosomes derived from GCs had similar miRNA profiles and follicle-promoting functions as follicular fluid exosomes. Consequently, GC-Exos are promising for replacing FF-Exos and developing new commercial reagents to improve female fertility.     

Circular RNA RSF1 promotes inflammatory and fibrotic phenotypes of irradiated hepatic stellate cell by modulating miR-146a-5p

The role of circular RNA (circRNA) in radiation-induced liver disease (RILD) remains largely unknown. In this study, Ras-related C3 botulinum toxin substrate 1 (RAC1) was elevated in irradiated human hepatic stellate cell (HSC) line LX2, the important effector cell mediating RILD. Overexpression of RAC1 promotes cell proliferation, proinflammatory cytokines production, and α-smooth muscle actin expression, which were blocked by microRNA (miR)-146a-5p mimics. CircRNA RSF1 (circRSF1) was upregulated in irradiated LX2 cells and predicted to harbor binding site for miR-146a-5p. Biotinylated-RNA pull down and dual-luciferase reporter detection confirmed the direct interaction of circRSF1 and miR-146a-5p. Enforced expression of circRSF1 increased RAC1 expression by acting as miR-146a-5p sponge to inhibit miR-146a-5p activity, and thus enhanced the cell viability, and promoted inflammatory and fibrotic phenotype of irradiated LX2 cells. These findings indicate a functional regulatory axis composing of circRSF1, miR-146a-5p, and RAC1 in irradiated HSC, which may provide attractive therapeutic targets for RILD.     

Mir-484 contributes to diminished ovarian reserve by regulating granulosa cell function via YAP1-mediated mitochondrial function and apoptosis

Women with diminished ovarian reserve (DOR) have reduced fertility, but the underlying regulation of ovarian function remains unknown. Although differential microRNA (miRNA) expression has been described in several ovarian disorders, little is known about the role of miRNAs in the pathogenesis of DOR. In this study, we investigated the expression levels of miR-484 in granulosa cells (GCs) derived from human follicular fluid, and explored their correlation with female ovarian reserve function as well as clinical outcomes of assisted reproduction technology (ART). Additionally, we investigated the effects of miR-484 on the biological functions of GC cell lines in vitro. We found that miR-484 was highly expressed in GCs from DOR patients and was correlated with decreasing AMH levels and AFC, as well as increasing FSH levels, but not with LH, progesterone, or estradiol. Additionally, miR-484 was negatively related to the number of retrieved oocytes and the ratio of high-quality embryos. Moreover, we found that miR-484 repressed the proliferation of GCs and induced apoptosis, which can in part be attributed to mitochondrial dysfunction. Conversely, silencing miR-484 had the opposite effect. Multiple approaches, including bioinformatic analysis, RNA-seq, qPCR, immunofluorescence, western blotting and luciferase reporter assays, identified YAP1 as a direct target of miR-484 in GCs. Additionally, reintroduction of YAP1 rescued the effects of miR-484 in GCs. The present study indicates that miR-484 can directly target the mRNA of YAP1, induce mitochondrial dysfunction, and consequently reduce the viability and promote the apoptosis of granulosa cells, which contributes to the pathogenesis of DOR.     

Expression profiles of key candidate genes involved in steroidogenesis during follicular atresia in the pig ovary

More than 99?% of follicles in mammalian ovaries undergo a degenerative process known as atresia, and thus only a limited number of ovarian follicles actually ovulate after full growth and development. The endocrinological regulatory mechanisms involved in follicular development have been studied extensively, but the precise and systematic molecular mechanisms of steroidogenesis enzymes involved in atresia are unclear. In the present study, we examined whether and how the steroidogenesis enzymes are involved in porcine ovary follicular atresia. Expression of steroidogenic acute regulatory protein, CYP11, CYP17, 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP19, as well as related pituitary and ovarian hormone receptors were quantified in ovaries. During porcine follicular atresia, expressions of P450 cholesterol side chain cleavage enzyme, progesterone and androgen receptors increased significantly during the late atretic stage, while the expression of aromatase and follicle-stimulating hormone receptors decreased significantly in the early stage. These data suggested that the regulation of aromatase by follicle-stimulating hormone might induce follicular atresia, and that progesterone and androgen production further promoted follicular atresia. Additionally, a correlation analysis indicated a large and complex interactive network among these genes and the endocrinological microenvironment of the follicles. Significant correlations were observed between expression of steroidogenic enzymes and their receptors, and also between progesterone and 17β-estradiol (E2) levels in follicular fluid. Taken together, these results suggest that CYP19 plays a role during early atresia by regulating the production of E2, whereas CYP11 and 3β-HSD increase atresia progression by increasing progesterone levels.     

Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells

The first objective of this study was to determine whether the death of bovine granulosa cells (GC) isolated from small ( 8 mm) follicles during follicular atresia occurs by apoptosis. The second objective was to establish an in vitro model system to elucidate the developmental (GC from follicles of different sizes) and hormonal (FSH and insulin-like growth factor-I [IGF-I]) regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric criteria as healthy or atretic. Apoptosis in GC from follicles of different sizes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 x 10(6) cells/ml in serum-free media at 39 degrees C to determine the effects of FSH and IGF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicles. Apoptosis in GC was also detected in some healthy follicles. Degenerate GC displayed the morphological characteristics of apoptosis, including nuclei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments, and/or membrane-bound structures containing variable amounts of chromatin and/or cytoplasm (apoptotic bodies). All GC classified as apoptotic on the basis of their morphology contained fragmented DNA measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the GC layer, apoptotic cell death was more prevalent among antral GC than among mural GC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in GC from small, medium, and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of GC from small follicles was higher than that from medium and large follicles. FSH attenuated apoptotic cell death in GC from medium follicles more effectively than in those from small follicles. IGF-I also suppressed apoptosis in cultured GC from small follicles. In conclusion, this study showed that 1) GC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in GC and theca cells; however, apoptosis does not occur in cumulus cells even in atretic antral follicles; 3) GC from all small, medium, and large follicles undergo spontaneous onset of apoptosis when cultured under serum-free conditions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC.     

The lncRNA SNHG15/miR-18a-5p axis promotes cell proliferation in ovarian cancer through activating Akt/mTOR signaling pathway

Here, we report the expression pattern, function and regulatory mechanism of SNHG15 together with miR-18a-5p micro RNA in ovarian cancer (OC) for the first time. We recruited 20 patients and took normal ovarian tissues and ovarian tumor tissues from them. We used cell culture, transfection, in vivo tumor xenograft assay, and multiple types of detection assays to investigate the expression and regulation of long noncoding RNA (lncRNA) SNHG15/miR-18a-5p in ovarian tissues and cells. Results: We found that the messenger RNA expression level of SNHG15 was significantly higher and miR-18 was decreased in ovarian cancer tissues and in OC cells. Functional experiments showed that SNHG15 overexpression potentiated the migration and invasion of OC cells, while SNHG15 inhibition reduced the tumor proliferation, which was restored via overexpression of miR-18a. SNHG15 was found to directly target and suppress the expression of miR-18a. Our results illustrate the possible molecular mechanism of lncRNA SNHG15/miR-18a-5p functions in cell proliferation in OC. SNHG15/miR-18a promoted the progression of OC cells via the protein kinase B/mammalian target of rapamycin signaling pathway.     

Down-regulation of miR-20a-5p triggers cell apoptosis to facilitate mycobacterial clearance through targeting JNK2 in human macrophages

Induction of cell apoptosis is one of the major host defense mechanisms through which macrophages control Mycobacterium tuberculosis (Mtb) infection. However, the mechanisms underlying macrophage apoptosis triggered by Mtb infection are still largely unknown. In this study, a microarray profiling survey revealed 14 miRNAs were down-regulated in CD14+ monocytes from active pulmonary tuberculosis patients, and only the reduction of miR-20a-5p could be reversed after successful anti-tuberculosis treatment. Validation of miR-20a-5p expression was confirmed using real time qPCR. Moreover, miR-20a-5p expression also decreased in differentiated THP-1 macrophages after mycobacterial infection in vitro. Functional assays through forced or inhibited expression of miR-20a-5p in THP-1 macrophages demonstrated that miR-20a-5p functioned as a negative regulator of mycobacterial-triggered apoptosis. Importantly, inhibition of miR-20a-5p expression resulted in more efficient mycobacterial clearance from infected THP-1 macrophages while miR-20a-5p overexpression promoted mycobacterial survival. Mechanistically, miR-20a-5p was demonstrated to regulate Bim expression in a JNK2-dependent manner, unlike Bcl2, and luciferase assay showed JNK2 was a novel direct target of miR-20a-5p. Together, our findings indicate that downregulation of miR-20a-5p triggers macrophage apoptosis as a novel mechanism for host defense against mycobacterial infection.     

Role of gelatinase on follicular atresia in the bovine ovary

Follicular atresia, like follicular growth and ovulation, is characterized by excessive tissue remodeling. It is hypothesized that probably one of the tissue-remodeling enzymes, such as the gelatinases, could be playing an important role in this process. The present study was undertaken to determine the role of gelatinase on follicular atresia in the cow. Follicles of 2-6 mm in diameter were dissected from ovaries, and follicular fluid was categorized according to the morphological appearance of the cumulus-oocyte complexes. Gelatinase activity within the follicular fluid was analyzed by gelatin zymography, and film in situ zymography was employed in order to localize gelatinase. TUNEL was performed on cryosectioned ovaries to understand follicular health. The concentrations of steroids in follicular fluid were also measured by solid phase fluoroimmunoassay. ProMMP-2 was detected in all normal and atretic categories of follicular fluid. The active form of MMP-2 and an additional band of proMMP-9 were detected only in atretic follicular fluid. Gelatinase activity was recorded in both granulosa cells (GCs) and theca cells (TCs) but were found in comparatively higher numbers in those follicles that exhibited a thinned and partially detached granulosa layer. TUNEL confirmed that apoptosis had commenced in the GCs of follicles of the latter category. The estradiol-17beta (E(2)):progesterone (P(4)) ratio was found to be significantly lower in atretic follicles than in normal follicles. These results suggest a plausible role for gelatinase in follicular health, especially the active form of MMP-2 and proMMP-9, and that bovine follicular fluid may be a key indicator of atresia.     

MicroRNA-19a-3p promotes rheumatoid arthritis fibroblast-like synoviocytes via targeting SOCS3

Rheumatoid arthritis (RA) is a common chronic autoimmune disease and effective treatment for RA is still lacking. In this study, the regulatory role of miR-19a-3p in RA was investigated. Quantitative polymerase chain reaction analysis of human blood samples showed that the level of miR-19a-3p was significantly lower in the RA patients compared with that in healthy patients (P < 0.05). In RA fibroblast-like synoviocytes (RAFLS), miR-19a-3p and suppressor of cytokine signaling 3 (SOCS3) were also downregulated and upregulated, respectively, compared with those of normal FLS. Transfection of miR-19a-3p mimic in RAFLS inhibited cell proliferation and promoted cell apoptosis. TargetScan identified SOCS3 as a target of miR-19a-3p, which was confirmed by dual-luciferase assay. Western blot indicated that SOCS3 protein level was significantly decreased after miR-19a-3p overexpression. Moreover, SOCS3 silencing through siRNA transfection also enhanced cell proliferation, meanwhile inhibiting RAFLS apoptosis. In addition, SOCS3 overexpression abrogated the effects of miR-19a-3p overexpression on cell proliferation and apoptosis, corroborating that SOCS3 acts as a downstream effector in the miR-19a-3p-mediated function of RAFLS. These findings suggest that miR-19a-3p plays an important role in RA, and the miR-19a-3p/SOCS3 axis may become a potential therapeutic target for RA.     

Circ-ERBB2 knockdown sensitized colorectal cancer cells to 5-FU via miR-181a-5p/PTEN/Akt pathway

Colorectal cancer (CRC) is the fourth most deadly cancer worldwide, drug resistance impedes treatment of CRC. It is still urgent to find new molecular targets to improve the sensitivity of chemotherapeutic drugs. In this study, circ-ERBB2 was upregulated in CRC cells. Upregulation of circ-ERBB2 promoted CRC cells proliferation and clone formation, but inhibited apoptosis. We identified miR-181a-5p as circ-ERBB2's target. The effect of miR-181a-5p on CRC cells was contrary to circ-ERBB2, miR-181a-5p downregulation abolished the function of circ-ERBB2 silencing in CRC cells. In addition, phosphatase and tensin homolog (PTEN) was verified as miR-181a-5p's downstream target, circ-ERBB2 activates the Akt pathway and inhibits cell apoptosis through modulating miR-181a-5p/PTEN. Circ-ERBB2 silencing significantly reduced CRC cell resistance to 5-FU. miR-181a-5p downregulation abolished the role of circ-ERBB2 knockdown in CRC cell resistance to 5-FU. In conclusion, upregulation of circ-ERBB2 promoted the malignancy of CRC and reduced CRC cell resistance to 5-FU. Besides, additional mechanism study provided a novel regulatory pathways that circ-ERBB2 knockdown promoted CRC cell sensitivity to 5-FU by regulating miR-181a-5p/PTEN/Akt pathway. This research indicated that circ-ERBB2 may be a valuable biomarker for the diagnosis and treatment of CRC.