RESUMO
The accumulation of ovarian granulosa cell (GC) apoptosis underlies follicular atresia. By comparing the previous sequencing results, miR-486 was found to be differentially expressed at higher levels in the monotocous goat than in the polytocous goat. Unfortunately, the miRNA-mediated mechanisms by which the GC fate is regulated are unknown in Guanzhong dairy goats. Therefore, we investigated miR-486 expression in small and large follicles, as well as its impact on normal GC survival, apoptosis and autophagy in vitro. Here, we identified and characterized miR-486 interaction with Ser/Arg-rich splicing factor 3 (SRSF3) using luciferase reporter analysis, detecting its role in GC survival, apoptosis and autophagy regulation through qRT-PCR, Western blot, CCK-8, EdU, flow cytometry, mitochondrial membrane potential and monodansylcadaverine, etc. Our findings revealed prominent effects of miR-486 in the regulation of GC survival, apoptosis and autophagy by targeting SRSF3, which might explain the high differential expression of miR-486 in the ovaries of monotocous dairy goats. In summary, this study aimed to reveal the underlying molecular mechanism of miR-486 regulation on GC function and its effect on ovarian follicle atresia in dairy goats, as well as the functional interpretation of the downstream target gene SRSF3.
Assuntos
Atresia Folicular , MicroRNAs , Animais , Feminino , Atresia Folicular/genética , Células da Granulosa/metabolismo , MicroRNAs/metabolismo , Apoptose/genética , Cabras/fisiologia , Autofagia/genéticaRESUMO
Paired-like homeodomain transcription factor 2 (PITX2), a major driver of multiple tissue development, is a transcription factor that regulates gene expression in organisms. However, it is unknown if PITX2 regulates goat granulosa cell (GC) steroidogenesis. Therefore, we investigated the role and mechanism of PITX2 in GC steroidogenesis. In our study, PITX2 significantly facilitated the secretion level of estrogen and progesterone through increasing CYP11A1, CYP19A1, and STAR mRNA and protein expressions in GCs. Furthermore, PITX2 participated in the WNT pathway, enhancing the production of E2 and P4 in GCs. PITX2 in GCs increased the DVL-1 and CTNNB1 expression, involved in the WNT/ß-catenin signaling pathway related to steroidogenesis. Moreover, GC steroidogenesis-related gene translation was decreased by CTNNB1-siRNA but enhanced when transfected with PITX2. PITX2 regulates secretion of E2 and P4 from GCs via the WNT/ß-catenin pathway and alters GC proliferation and steroidogenesis. These findings will help understand the role of PITX2 in goat ovarian follicular development and oocyte maturation.
Assuntos
Via de Sinalização Wnt , beta Catenina , Animais , Feminino , Cabras/metabolismo , Células da Granulosa/metabolismo , Fatores de Transcrição/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Human induced pluripotent stem cell (hiPSC)-derived mesenchymal stem cells (iMSCs) are ideal candidates for the production of standardised and scalable bioengineered bone grafts. However, stable induction and osteogenic differentiation of iMSCs pose challenges in the industry. We developed a precise differentiation method to produce homogeneous and fully differentiated iMSCs. In this study, we established a standardised system to prepare iMSCs with increased osteogenic potential and improved bioactivity by introducing a CHIR99021 (C91)-treated osteogenic microenvironment (COOME). COOME enhances the osteogenic differentiation and mineralisation of iMSCs via canonical Wnt signalling. Global transcriptome analysis and co-culturing experiments indicated that COOME increased the pro-angiogenesis/neurogenesis activity of iMSCs. The superior osteogenic differentiation and mineralisation abilities of COOME-treated iMSCs were also confirmed in a Bio3D module generated using a polycaprolactone (PCL) and cell-integrated 3D printing (PCI3D) system, which is the closest model to in vivo research. This COOME-treated iMSCs differentiation system offers a new perspective for generating highly osteogenic, bioactive, and anatomically matched grafts for clinical applications. Statement of significance: Although human induced pluripotent stem cell-derived MSCs (iMSCs) are ideal seed cells for synthetic bone implants, the challenges of stable induction and osteogenic differentiation hinder their clinical application. This study established a standardised system for the scalable preparation of iMSCs with improved osteogenic potential by combining our precise iMSC differentiation method with the CHIR99021 (C91)-treated osteocyte osteogenic microenvironment (COOME) through the activation of canonical Wnt signalling. Moreover, COOME upregulated the pro-angiogenic and pro-neurogenic capacities of iMSCs, which are crucial for the integration of implanted bone grafts. The superior osteogenic ability of COOME-treated iMSCs was confirmed in Bio3D modules generated using PCL and cell-integrated 3D printing systems, highlighting their functional potential in vivo. This study contributes to tissue engineering by providing insights into the functional differentiation of iMSCs for bone regeneration.
RESUMO
Human and animal exposure to microplastics (MPs) contained in food is inevitable because of their widespread existence in the environment. Nevertheless, MPs toxicity studies in ruminants often lack attention. Here, we assessed the cytotoxicity of polystyrene microplastics (PS MPs) on goat mammary epithelial cells (GMECs). Compared to controls, PS MPs treatment significantly reduced cell viability, altered cell morphology and disrupted organelle integrity. Detection of membrane potential and reactive oxygen species (ROS) suggested that PS MPs induced mitochondrial dysfunction and oxidative stress. Further transcriptome analysis also confirmed alterations in these pathways. In addition, several genes related to endoplasmic reticulum (ER) homeostasis were significantly regulated in the transcriptional profile. Subsequent experiments confirmed that PS MPs induce ER stress via the PERK/eIF2α/CHOP pathway, accompanied by intracellular Ca2+ overload. Meanwhile, downstream activation of the Bax/Bcl-2 pathway and caspase cascade released apoptotic signals, which led to apoptosis in GMECs. Interestingly, the addition of PERK inhibitor (ISRIB) attenuated PS MPs-induced ER stress and apoptosis, which suggests that ER stress may exacerbate PS MPs-induced cytotoxicity. This work reveals the impact of MPs on mammalian cytotoxicity, enriches the mechanisms for the toxicity of MPs, and provides insight for further assessment of the risk of MPs in food.
Assuntos
Microplásticos , Plásticos , Animais , Humanos , Microplásticos/toxicidade , Microplásticos/metabolismo , Estresse do Retículo Endoplasmático , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Mamíferos/metabolismoRESUMO
Nrf1 (encoded by Nfe2l1) and Nrf2 (encoded by Nfe2l2), as two key members of the CNC-bZIP transcription factor, exhibit significant functional differences in their pathophysiology. Our previous findings demonstrated that loss of Nrf1α (i.e., a full-length isoform of Nrf1) promotes HepG2-derived tumor growth in xenograft mice, but malgrowth of the xenograft tumor is significantly suppressed by knockout of Nrf2. To gain insights into the mechanism underlying such marked distinctions in their pathologic phenotypes, we mined transcriptome data from liver cancer in the TCGA database to establish a prognostic model and calculate predicted risk scores for each cell line. The results revealed that knockout of Nrf1α markedly increased the risk score in HepG2 cells, whereas the risk score was reduced by knockout of Nrf2. Notably, stanniocalcin 2 (STC2), a biomarker associated with liver cancer, that is upexpressed in hepatocellular carcinoma (HCC) tissues with a reduction in the overall survival ratio of those patients. We observed increased expression levels of STC2 in Nrf1α-/- cells but decreased expression in Nrf2-/- cells. These findings suggested that STC2 may play a role in mediating the distinction between Nrf1α-/- and Nrf2-/-. Such potential function of STC2 was further corroborated through a series of experiments combined with transcriptomic sequencing. The results revealed that STC2 functions as a dominant tumor-promoter, because the STC2-leading increases in clonogenicity of hepatoma cells and malgrowth of relevant xenograft tumor were almost completely abolished in STC2-/- cells. Together, these demonstrate that STC2 could be paved as a potential therapeutic target, albeit as a diagnostic marker, for HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animais , Camundongos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Fator 2 Relacionado a NF-E2/genética , Peptídeos e Proteínas de Sinalização Intercelular , Biomarcadores , Glicoproteínas/genética , Glicoproteínas/metabolismoRESUMO
PTX3, a member of the pentraxin protein family, plays important roles in ovulation as a marker of cumulus cell-oocyte complex expansion. However, the expression and function of PTX3 in goat ovarian GCs remain unclear. We isolated GCs from small and large follicles and found that PTX3 expression was significantly decreased and miR-29 mRNA expression was significantly increased during the growth of antral follicles. MiR-29 decreased PTX3 expression by targeting its 3' untranslated. Furthermore, miR-29 promoted GC proliferation, suppressed steroidogenesis and apoptosis by targeting PTX3 via the activation of the PI3K/AKT/mTOR and Erk1/2 signaling pathways. Treatment with inhibitors also verified these results. Meanwhile, we found that PI3K/AKT/mTOR and Erk1/2 signaling pathways had different role in secretion of E2 and P4 by regulating differently various steroidogenic enzyme (CYP19A1, CYP11A1, StAR and HSD3B) expression. These outcomes indicate the potential role of PTX3 in goat follicular growth and atresia.