Aug 2026· Genes· Vol 17, pp. 966· 0 citations· 38 references
Medicine
TL;DR
A novel pathogenic mechanism whereby miR-27b-3p exacerbates VCD-induced granulosa cell injury by directly targeting PAPPA, suppressing IGF-1 release, and consequently inhibiting the PI3K/AKT pro-survival signaling pathway is uncovered.
Abstract
Background/Objectives: While currently termed premature ovarian insufficiency (POI), premature ovarian failure (POF) remains a prominent driver of female infertility with a molecular pathogenesis that is still lacking comprehensive clarification. In in vitro studies, the pathology of POI is commonly simulated through a well-characterized model involving VCD (4-vinylcyclohexene diepoxide)-mediated cytotoxicity within KGN-derived human granulosa cells. However, the key regulatory molecular networks involved in this process are still poorly characterized. Although microRNAs (miRNAs) have emerged as critical regulators in ovarian function decline, the specific role and underlying mechanism of miR-27b-3p in POI remain elusive. Methods: A VCD-induced KGN cell injury model was established by treating cells with 1.0 mM VCD for 24 h. Cell viability, apoptosis rate, and miR-27b-3p expression were assessed by CCK-8 assay, flow cytometry, and RT-qPCR, respectively. Overexpression and targeted suppression of miR-27b-3p were achieved by introducing its specific mimics and inhibitors, respectively. Target identification was conducted via bioinformatic prediction, EdU incorporation, Western blot, and dual-luciferase reporter assays. Functional rescue experiments were carried out by co-transfection with a PAPPA-overexpressing plasmid (oe-PAPPA). IGF-1 secretion was quantified by ELISA, and phosphorylation of IGF1R and AKT was analyzed by Western blot to determine whether miR-27b-3p modulates cellular phenotypes via the PAPPA–IGF-1–PI3K/AKT axis. Exogenous IGF-1 supplementation was further applied to confirm pathway dependence. Results: VCD treatment dose-dependently restrained cellular growth and stimulated apoptotic pathways in KGN cells; paralleling these phenotypic changes, miR-27b-3p abundance was remarkably increased. Ectopic expression of miR-27b-3p exacerbated VCD-induced growth inhibition and apoptosis, whereas its inhibition conferred cytoprotective effects. Through the integration of computational predictions and dual-luciferase reporter systems, PAPPA was definitively established as a direct downstream target of miR-27b-3p. miR-27b-3p negatively regulated both PAPPA mRNA and protein levels, thereby impairing PAPPA-mediated cleavage of IGF-binding proteins (e.g., IGFBP4) and subsequent release of free IGF-1. This led to reduced IGF-1 secretion and significantly diminished phosphorylation of IGF1R and AKT. Remarkably, PAPPA overexpression effectively reversed the detrimental effects of miR-27b-3p, and exogenous IGF-1 supplementation similarly attenuated miR-27b-3p–mediated proliferation arrest and pro-apoptotic phenotypes. Conclusions: This study uncovers a novel pathogenic mechanism whereby miR-27b-3p exacerbates VCD-induced granulosa cell injury by directly targeting PAPPA, suppressing IGF-1 release, and consequently inhibiting the PI3K/AKT pro-survival signaling pathway. A novel perspective on the fundamental basis of POI is established by this research, which further posits therapeutic manipulation of the miR-27b-3p/PAPPA/IGF-1 module as a prospective treatment for disrupted ovarian function.
Cryptorchidism, among the most frequent congenital urogenital anomalies in male infants, has a global prevalence ranging from 1% to 9%. Characterized by failure of testicular descent into the scrotum, such a condition exposes the testes to elevated temperature, oxidative stress, and impaired spermatogenesis, significantly increasing risks of infertility and testicular cancer. Although orchiopexy is the standard treatment, long-term spermatogenic defects often persist, highlighting the need to elucidate underlying molecular mechanisms. Although the main mechanistic experiments were performed in GC-1 spg cells through genetic manipulation of cPKCγ, an additional heat-stress experiment was included to simulate the high-temperature condition associated with cryptorchidism. In the present study, the role of conventional protein kinase C gamma (cPKCγ) in regulating mitophagy and apoptosis was investigated using mouse spermatogonial GC-1 cells. Through siRNA-mediated knockdown and adenovirus-induced overexpression, cPKCγ upregulation was demonstrated to significantly induce cellular apoptosis and disrupt mitochondrial function, indicated by elevated malondialdehyde (MDA) levels, reduced superoxide dismutase (SOD) activity, decreased ATP production, increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) accumulation, increased mitochondrial reactive oxygen species (ROS), and loss of mitochondrial membrane potential. Mechanistically, cPKCγ was found to inhibit the UCHL1/HIF-1α signaling pathway, suppressing mitophagy, as evidenced by a reduced LC3-II/LC3-I ratio and decreased PINK1 and Parkin expression. Importantly, adverse effects induced by cPKCγ overexpression were substantially rescued through UCHL1 or HIF-1α overexpression, or rapamycin treatment. Conversely, knockdown of UCHL1 or HIF-1α partially counteracted the protective effects of cPKCγ silencing on cell viability, oxidative stress, and mitophagy activation. Furthermore, heat stress upregulated endogenous cPKCγ and suppressed UCHL1/HIF-1α signaling and mitophagy-related proteins in GC-1 spg cells. Collectively, cPKCγ exacerbates spermatogonial apoptosis by repressing UCHL1/HIF-1α-dependent mitophagy, suggesting a potential molecular mechanism for spermatogonial vulnerability under cryptorchidism-related heat stress.
Huajun Yang, You-You Tian, Shaoguang Feng et al.· Systems Biology in Reproduct...· 0 citations
Background Diminished ovarian reserve (DOR) is a common cause of female infertility. Prior high-throughput sequencing showed specific follicular fluid exosomal miRNA profiles in DOR patients, implicating exosomal miRNAs in DOR pathogenesis. Using miRNA PCR arrays, we found miR-483-3p was significantly upregulated in DOR follicular fluid exosomes. Bioinformatics and experiments indicated METTL3 as a potential miR-483-3p target, suggesting excess miR-483-3p exerts cytotoxicity and suppresses METTL3-mediated N6-methyladenosine (m6A) modification. This study thus investigated miR-483-3p effects on METTL3 expression, m6A levels, cell proliferation, and mitochondrial function in ovarian granulosa cells (GCs). Methods The targeting interaction between miR-483-3p and METTL3 was confirmed using a dual-luciferase reporter gene assay. qRT-PCR, immunofluorescence, Western blot, and Dot blot were used to evaluate the effects of miR-483-3p mimics, cyclophosphamide (CTX), and STM2457 on METTL3 protein expression and m6A modification levels in KGN cells. Additionally, Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2’-deoxyuridine (EdU) assays, along with mitochondrial membrane potential (MMP) measurements, were used to examine the impact of these treatments on KGN cell proliferation and mitochondrial function. Results miR-483-3p directly targeted the 3′ untranslated region (3′UTR) of METTL3, and its mimics significantly suppressed METTL3 gene expression. In contrast, CTX robustly promoted METTL3 gene expression, accompanied by a substantial elevation in global m6A levels. STM2457 treatment also showed a similar trend. But miR-483-3p overexpression modestly inhibited METTL3 protein and m6A levels. Regarding cellular phenotypes, miR-483-3p overexpression, CTX, and STM2457 all exerted significant inhibitory effects on KGN cells. All three treatments consistently suppressed the cell viability of KGN cells, reduced the EdU-positive cell ratio, and decreased MMP levels. Conclusion This study provides preliminary evidence suggesting a specific regulatory interaction between miR-483-3p and METTL3. We demonstrate that aberrantly high expression of miR-483-3p significantly suppresses METTL3 mRNA levels but only moderately reduces METTL3 protein and m6A levels, suggesting the involvement of complex post-transcriptional regulatory mechanisms. Furthermore, overaccumulation of miR-483-3p markedly inhibits the proliferation of ovarian GCs and impairs their mitochondrial function. Further investigation into the role of the candidate miR-483-3p-METTL3-m6A axis in DOR will be conducted using in vivo models alongside an expanded cohort of clinical samples.
Combination therapies targeting multiple of these glucose signaling pathway proteins, together with glucose and N-acetylcysteine, yielded superior therapeutic benefit in complex I disease cell and C. elegans models.
Kelsey Keith, Min Peng, Cristina Remes et al.· bioRxiv· 0 citations
BACKGROUND
To investigate the protective effect of M2 macrophage-derived exosomes (M2-Exo) on high glucose-induced podocyte injury and the underlying molecular mechanism.
METHODS
THP-1 cells were induced to differentiate into M2 macrophages using PMA combined with IL-4/IL-13, and exosomes were extracted and identified. A high glucose-induced podocyte injury model was established in vitro, and a streptozotocin (STZ)-induced diabetic nephropathy (DN) mouse model was established in vivo to evaluate the protective effect and mechanism of M2-Exo.
RESULTS
M2-Exo were successfully isolated and identified. M2-Exo intervention restored podocyte viability, inhibited apoptosis, and suppressed NLRP3-mediated pyroptosis. SET-binding factor 2-antisense RNA 1 (SBF2-AS1) was highly expressed in M2-Exo and functioned as a competing endogenous RNA (ceRNA) by competitively binding to miR-650, thereby upregulating mouse double minute 2 (MDM2) expression. MDM2, acting as an E3 ubiquitin ligase, promoted NIMA-related kinase 7 (NEK7) ubiquitination and degradation, subsequently inhibiting NOD-like receptor family pyrin domain containing 3 (NLRP3) pyroptosis pathway activation. In vivo experiments confirmed that SBF2-AS1-overexpressing M2-Exo improved renal function and alleviated renal pathological injury in DN mice.
CONCLUSION
M2-Exo deliver SBF2-AS1 to regulate the miR-650/MDM2/NEK7 axis, inhibit NLRP3-mediated podocyte pyroptosis, and ameliorate the progression of DN.
Yan Zhang, Qing Zhan, Min Zhao et al.· Cellular Signalling· 0 citations
Colorectal cancer (CRC) is a prevalent malignancy worldwide, with its immune evasion representing a pivotal barrier to effective immunotherapy. OLR1 is abnormally expressed in a variety of cancers, but its function in the CRC immune microenvironment and the upstream and downstream modulatory mechanisms remain unclear. Based on the TCGA-COAD data set, OLR1 was upregulated in colorectal cancer (CRC) and correlated with CD8+ T-cell infiltration. Functional assays (qRT-PCR, Western blotting, LDH, ELISA, CCK-8, CFSE, colony formation, Transwell, and flow cytometry) confirmed that OLR1 suppressed CD8+ T-cell function and promoted tumor malignancy. GSEA and inhibitor-based functional validation identified key pathways downstream of OLR1. Mechanistically, bioinformatics, RIP, RNA pull-down, MeRIP, and actinomycin D assays revealed that IGF2BP3 stabilized OLR1 mRNA. Rescue experiments further demonstrated that the IGF2BP3/OLR1 axis regulated the aforementioned pathways and CRC immune evasion. OLR1 was upregulated in CRC and negatively associated with CD8+ T-cell infiltration. Knockdown of OLR1 significantly enhanced the antitumor function of CD8+ T cells. Mechanistically, IGF2BP3 stabilized OLR1 mRNA and up-regulated its expression in an m6A modification-dependent manner, thereby activating the PI3K/AKT/mTOR pathway to inhibit the antitumor activity of CD8+ T cells. IGF2BP3 stabilized OLR1 through m6A modification and activated the PI3K/AKT/mTOR pathway, thereby repressing the antitumor activity of CD8+ T cells and promoting immune evasion in CRC.
