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As apple fruits (Malus domestica) mature, they accumulate anthocyanins concomitantly with losing chlorophyll (Chl); however, the molecular pathways and events that coordinate Chl degradation and fruit coloration have not been elucidated. We showed previously that the transcription factor ETHYLENE RESPONSE FACTOR17 (MdERF17) modulates Chl degradation in apple fruit peels and that variation in the pattern of MdERF17 serine (Ser) residues is responsible for differences in its transcriptional regulatory activity. Here, we report that MdERF17 interacts with and is phosphorylated by MAP KINASE4 (MdMPK4-14G). Phosphorylation of MdERF17 at residue Thr67 by MdMPK4-14G is necessary for its transcriptional regulatory activity and its regulation of Chl degradation. We also show that MdERF17 mutants with different numbers of Ser repeat insertions exhibit altered phosphorylation profiles, with more repeats increasing its interaction with MdMPK4. MdMPK4-14G can be activated by exposure to darkness and is involved in the dark-induced degreening of fruit peels. We also demonstrate that greater phosphorylation of MdERF17 by MdMPK4-14G is responsible for the regulation of Chl degradation during light/dark transitions. Overall, our findings reveal the mechanism by which MdMPK4 controls fruit peel coloration.

Follicular atresia triggered by granulosa cell (GC) apoptosis severely reduces female fertility and accelerates reproductive aging. GC apoptosis is a complex process regulated by multiple factors, regulatory axes, and signaling pathways. Here, we report a novel, small regulatory network involved in GC apoptosis and follicular atresia. miR-187, a miRNA down-regulated during follicular atresia in sows, maintains TGFBR2 mRNA stability in sow GCs by directly binding to its 5’-UTR. miR-187 activates the transforming growth factor-β (TGF-β) signaling pathway and suppresses GC apoptosis via TGFBR2 activation. NORHA, a pro-apoptotic lncRNA expressed in sow GCs, inhibits TGFBR2-mediated activation of the TGF-β signaling pathway by sponging miR-187. In contrast, NORFA, a functional lncRNA associated with sow follicular atresia and GC apoptosis, enhances miR-187 and TGFBR2 expression by inhibiting NORHA and activating NFIX. Our findings define a simple regulatory network that controls GC apoptosis and follicular atresia, providing new insights into the mechanisms of GC apoptosis, follicular atresia, and female fertility.

Deep-sea cold seeps are one of the most productive ecosystems that sustained by hydrocarbons carried by the fluid. Once the seep fluid ceases, the thriving autotrophic communities die out, terming as the extinct seep. But heterotrophic fauna can still survive even for thousands of years. The critical role of prokaryotes in active seeps are well defined, but their functions in extinct seeps are poorly understood to date. Here, we clarified the diversity, taxonomic specificity, interspecies correlation, and metabolic profiles of sediment prokaryotes at an extinct seep site of Haima cold seep, South China Sea. Alpha diversity of archaea significantly increased, while that of bacteria remained unchanged in extinct seep compared to active seep. However, archaea composition did not differ significantly at extinct seep from active or nonseep sites based on weighted-unifrac dissimilarity, while bacteria composition exhibited significant difference. Distribution of archaea and bacteria showed clear specificity to extinct seeps, indicating the unique life strategies here. Prokaryotes might live chemolithoautotrophically on cycling of inorganic carbon, sulfur, and nitrogen, or chemoorganotrophically on recycling of hydrocarbons. Notably, many of the extinct seep specific species and networked keystone lineages are classified as Proteobacteria. Regarding the functional diversity and metabolic flexibility of this clade, Proteobacteria is supposed to integrate the geochemical cycles and play a critical role in energy and resource supplement for microbiome in extinct seep. Collectively, our findings shed lights on the microbial ecology and functional diversity in extinct seeps, providing new understanding of biogeochemical cycling after fluid cessation.

The bacterium Gordonia sp. SCSIO19801, which could effectively utilize phenanthrene as the sole carbon source, was isolated from the seawater of the South China Sea. Its biodegradation characteristics, whole genome sequence, and biodegradation pathway were investigated. The phenanthrene biodegradation process of Gordonia sp. SCSIO19801 was estimated to be a first-order kinetic model with a k value of 0.26/day. Based on the identification of metabolites, utilization of probable intermediates, and genomics analysis of related genes, the degradation of phenanthrene by Gordonia sp. SCSIO19801 was proposed to occur via the salicylate metabolic pathway. This is the first report of a phenanthrene degradation pathway in Gordonia species. In addition, the Gordonia sp. SCSIO19801 could use other aromatic compounds as the sole source of carbon and energy. These characteristics indicate that Gordonia sp. SCSIO19801 can be utilized for developing effective methods for the biodegradation of petroleum hydrocarbons in marine environments.

Transforming growth factor-β1 (TGF-β1) is essential for ovarian function and female fertility in mammals. Herein, we identified three completely linked variants, including two known variants referred to as c.1583A > G and c.1587A > G and the novel variant c.2074A > C in the porcine TGF-β1 3′-UTR. An important role of these variants in Yorkshire sow fertility was revealed. Variants c.1583A > G and c.1587A > G were located at the miRNA response element (MRE) of miR-2337 and affected miR-2337 regulation of TGF-β1 3′-UTR activity. Interestingly, miR-2337 induces, not reduces the transcription and production of TGF-β1 in granulosa cells (GCs). Mechanistically, miR-2337 enhances TGF-β1 promoter activity via the MRE motif in the core promoter region and alters histone modifications, including H3K4me2, H3K4me3, H3K9me2, and H3K9ac. In addition, miR-2337 controls TGF-β1-mediated activity of the TGF-β signaling pathway and GC apoptosis. Taken together, our findings identify miR-2337 as an endogenous small activating RNA (saRNA) of TGF-β1 in GCs, while miR-2337 is identified as a small activator of the TGF-β signaling pathway which is expected to be a new target for rescuing GC apoptosis and treating low fertility.

Transforming growth factor β1 (TGF-β1), an essential cytokine belongs to TGF-β superfamily, is crucial for female fertility. Increasing evidence show that long noncoding RNAs (lncRNAs) influence the state of granulosa cells (GCs). This study aimed to detect the effects of TGF-β1 on the lncRNA transcriptome, and investigate whether lncRNAs mediate the functions of TGF-β1 in GCs.RNA-seq and bioinformatics analyses were performed to identify and characterize the differentially expressed lncRNAs (DElncRNAs). The regulatory mechanism of TGF-β1 to lncRNA transcriptome was analyzed by chromatin immunoprecipitation. The effects of lncRNAs on the antiapoptotic and proproliferative functions of TGF-β1 were examined by morphological analysis, fluorescence-activated cell sorting, Cell Counting Kit-8, and Western blot.A total of 72 DElncRNAs highly sensitive to TGF-β1 were identified with the criteria of |logOur findings demonstrate that TGF-β1 alters lncRNA transcriptome in a SMAD4-dependent manner, and highlight that lncRNAs mediate the functions of TGF-β1 in GCs, which contribute to a better understanding of the epigenetic regulation of female fertility.

The progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) correlates with rupture of lysosome in Parkinson's disease (PD). It has been found that TP53-induced glycolysis and apoptosis regulator (TIGAR) has been attributed to the regulation of metabolic pathways and neuroprotective effect. In the present study, we showed in a mouse model that 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) caused lysosomal damage and DA neurons loss in the SNpc. MPTP only induced SP1-mediated TIGAR upregulation in the early stage of neurotoxin-induced pathology, and this compensatory mechanism was not enough to maintain normal lysosomal function. MPTP significantly decreased the levels of NADPH and GSH, and the effects were ameliorated by the expression of exogenous TIGAR but execerbated by knockdown of TIAGR. TIGAR or NADPH alleviated oxidative stress, rescued lysosomal dysfunction and attenuated DA neurons degeneration. Overexpression of TIGAR or NADPH supplement inhibited MPP+-mediated reactive oxygen species (ROS), lysosomal membrane permeabilization (LMP) and autophagic flux impairment in PC12 cells. Together, these findings suggest that TIGAR reduces MPTP-mediated oxidative stress, lysosomal depletion and DA neuron damage.

Increasing evidence shows that lncRNAs, an important kind of endogenous regulator, are involved in the regulation of follicular development and female fertility, but the mechanism remain largely unknown. In this study, we found that SDNOR, a recently identified antiapoptotic lncRNA, is a potential multifunctional regulator in porcine follicular granulosa cells (GCs) through RNA-seq and multi-dimension analyses. SDNOR-mediated regulatory networks were established and identified that SOX9, a transcription factor inhibited by SDNOR, mediates SDNOR’s regulation of the transcription of downstream targets. Functional analyses showed that loss of SDNOR significantly impairs GC morphology, inhibits cell proliferation and viability, reduces E2/P4 index, and suppresses the expression of crucial markers, including PCNA, Ki67, CDK2, CYP11A1, CYP19A1, and StAR. Additionally, after the detection of ROS, SOD, GSH-Px, and MDA, we found that SDNOR elevates the resistance of GCs to oxidative stress (OS) and also inhibits OS-induced apoptosis. Notably, GCs with high SDNOR levels are insensitive to oxidative stress, leading to lower apoptosis rates and higher environmental adaptability. In summary, our findings reveal the regulation of porcine GCs in response to oxidative stress from the perspective of lncRNA and demonstrate that SDNOR is an essential antioxidative lncRNA for maintaining the normal state and function of GCs.

TGF-β1, an important multi-functional cytokine of the TGF-β signaling pathway, has been reported to be crucial for ovarian granulosa cell (GC) states and female fertility. However, the molecular mechanism underlying TGF-β1 regulation of GC states remains largely unknown. Here, we provide a comprehensive transcriptomic view on TGF-β1 regulation of cell states in porcine GCs. We first confirmed that TGF-β1 can control GC states (apoptosis and proliferation) in pig ovary. RNA-seq showed that 909 differentially expressed genes (DEGs), including 890 DEmRNAs and 19 DEmiRNAs, were identified in TGF-β1-treated porcine GCs. Functional annotation showed that these DEGs were mainly involved in regulating cell states. In addition, multiple hub genes were identified by constructing the protein-protein interaction network, DEmiRNA-DEmRNAs regulatory network, and gene-pathway-function co-expression networks, which were further found to be enriched in FoxO, TGF-β, Wnt, PIK3-Akt, p53 and Ras signaling pathways that play important roles in regulating cell states, cell cycle, proliferation, stress-responses and inflammation. The current research deeply reveals the effects of TGF-β1 on porcine GCs, and also identifies potential therapeutic RNA molecules for inhibiting and rescuing female infertility.

TGF-β1 is a ligand of the TGF-β superfamily and an important cytokine that regulates ovarian functions including follicular development, steroid production, ovulation, luteinization, and female fertility. However, little is known about the regulation of TGF-β1 expression in ovary. Here, we identified that TGF-β1 is a functional target of miR-130a in porcine ovarian granulosa cells (GCs). The 3′-UTR sequence of TGF-β1 gene (1137 bp in length) in Large White (LW) pig was isolated, and multiple RNA regulatory elements (RREs), including several binding motifs of different miRNAs, were identified in this region. Luciferase activity assay showed that miR-130a dramatically suppresses the 3′-UTR luciferase activity of TGF-β1 gene, and further inhibits the expression of TGF-β1 in porcine GCs. FACS revealed that miR-130a acts as a pro-apoptotic factor and promotes GC apoptosis by inhibiting TGF-β1. Two novel linked mutations (−573G > A and −540T > C) were identified in the promoter region of ssc-miR-130a, but their polymorphisms are not associated with sow reproductive traits. Importantly, combined genotype analysis with a known mutation (c.1583 A > G) in the 3′-UTR of porcine TGF-β1 gene showed a significant association with reproductive performance in LW sow population. Overall, our findings defined a novel regulatory axis, miR-130a/TGF-β1 axis, which is involved in regulating sow fertility.

(E)-2-(2-(2,3-dibromo-4,5-dimethoxybenzylidene)hydrazinyl)-4-(3,4-difluorophenyl) thiazole (DDH-1) is novel small molecule compound synthesized in our previous work. This study found that DDH-1 could inhibit the proliferation of various human lung cancer cells. Particularly, the IC50 for A549 cells was 8.59 μM. Interestingly, we found that DDH-1 inhibits eIF4E/eIF4G interaction. The flow cytometry (FACS) results have indicated that DDH-1 may induce G0/G1 cycle arrest by inhibiting the expression of Cyclin D1 and CDK4. DDH-1 may also cause apoptosis through the caspase-dependent pathway. Study of these mechanisms has shown that DDH-1 may prompt reactive oxygen species (ROS) generation, decrease the mitochondrial membrane potential (MMP), and stimulate DNA double-strand breaks (DSBs) in A549 cells. Study of the signal pathway has indicated that DDH-1 could activate JNK phosphorylation and inhibit ERK phosphorylation. Interestingly, NAC, which scavenges ROS, reversed the MMP decline, DNA damage, JNK phosphorylation activation, and ERK phosphorylation inhibition caused by DDH-1. Overall, these findings provide evidence that the eIF4E/eIF4G interaction inhibitors DDH-1 induces DNA damage and apoptosis in human lung cancer A549 cells.

The TGF-β and Wnt signaling pathways are interrelated in many cell types and tissues, and control cell functions in coordination. Here, we report that SMAD4, a downstream effector of the TGF-β signaling pathway, induces FZD4, a receptor of the Wnt signaling pathway, establishing a novel route of communication between these two pathways in granulosa cells (GCs). We found that SMAD4 is a strong inducer of FZD4, not only initiating FZD4 transcription but also activating FZD4-dependent Wnt signaling and GC apoptosis. Furthermore, we identified the direct and indirect mechanisms by which SMAD4 promotes expression of FZD4 in GCs. First, SMAD4 functions as a transcription factor to directly bind to the FZD4 promoter region to increase its transcriptional activity. Second, SMAD4 promotes production of SDNOR, a novel lncRNA that acts as a sponge for miR-29c, providing another mean to block miR-29c from degenerating FZD4 mRNA. Overall, our findings not only reveal a new channel of crosstalk between the TGF-β and Wnt signaling pathways, SMAD4–FZD4 axis, but also provide new insights into the regulatory network of GC apoptosis and follicular atresia. These RNA molecules, such as miR-29c and lnc-SDNOR, represent potential targets for treatment of reproductive diseases and improvement of female fertility.

Long intergenic non-coding RNAs (lincRNAs) have been proved to be involved in regulating female reproduction. However, to what extent lincRNAs are involved in ovarian functions and fertility is incompletely understood. Here we show that a lincRNA, NORFA is involved in granulosa cell apoptosis, follicular atresia and sow fertility. We found that NORFA was down-regulated during follicular atresia, and inhibited granulosa cell apoptosis. NORFA directly interacted with miR-126 and thereby preventing it from binding to TGFBR2 3′-UTR. miR-126 enhanced granulosa cell apoptosis by attenuating NORFA-induced TGF-β signaling pathway. Importantly, a breed-specific 19-bp duplication was detected in NORFA promoter, which proved association with sow fertility through enhancing transcription activity of NORFA by recruiting transcription factor NFIX. In summary, our findings identified a candidate lincRNA for sow prolificacy, and provided insights into the mechanism of follicular atresia and female fertility., Xing Du et al. show that long intergenic non-coding RNA (lincRNA) NORFA maintains sow fertility by inhibiting apoptotic death of granulosa cells through the TGF-β signaling pathway. A breed-specific 19-bp duplication suggests that transcription factor NFIX may enhance the transcription of NORFA, providing insights into the role of lincRNAs for female fertility.

Maternal obesity is associated with placental oxidative stress. However, the mechanism underlying this association remains poorly understood. In the present study, a gilt obesity model was developed by exposure to different energy diets and used to investigate the role of NADPH oxidase 2 (Nox2) in the placenta. Specifically, 99 gilts (Guangdong Small-ear Spotted pig) at day 60 of gestation were randomly assigned to one of the following three treatments: low-energy group (L, DE=11.50 MJ/kg), medium-energy group (M, DE=12.41 MJ/kg), and high-energy group (H, DE=13.42 MJ/kg), with 11 replicate pens per treatment and 3 gilts per pen. At the start of the study, maternal body weight and backfat thickness were not significantly different in the three treatments. After the study, data indicated that the H group had higher body weight and backfat thickness gain for gilts during gestation and lower piglet birth weight compared with the other two groups. Additionally, the H group showed glucolipid metabolic disorders and increased triglyceride and nonesterified fatty acid contents in the placenta of gilts. Compared with the L group, the H group exhibited lower mitochondrial biogenesis and increased oxidative damage in the placenta. Importantly, increased mRNA expression and protein abundance of Nox2 were observed for the first time in H group placentae. Furthermore, compared with the L group, the H group showed a decrease in the density of placental vessels and the protein levels of vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor A (VEGF-A), and phosphorylation of vascular endothelial growth factor receptor 2 (p-VEGFR2) as well as the immunostaining intensity of platelet endothelial cell adhesion molecule-1 (CD31). Our findings suggest that maternal high-energy diet-induced obesity increases placental oxidative stress and decreases placental angiogenesis possibly through the upregulation of Nox2.

Summary: Insulin-stimulated hepatic glycogen synthesis is central to glucose homeostasis. Here, we show that PPP1R3G, a regulatory subunit of protein phosphatase 1 (PP1), is directly phosphorylated by AKT. PPP1R3G phosphorylation fluctuates with fasting-refeeding cycle and is required for insulin-stimulated dephosphorylation, i.e., activation of glycogen synthase (GS) in hepatocytes. In this study, we demonstrate that knockdown of PPP1R3G significantly inhibits insulin response. The introduction of wild-type PPP1R3G, and not phosphorylation-defective mutants, increases hepatic glycogen deposition, blood glucose clearance, and insulin sensitivity in vivo. Mechanistically, phosphorylated PPP1R3G displays increased binding for, and promotes dephosphorylation of, phospho-GS. Furthermore, PPP1R3B, another regulatory subunit of PP1, binds to the dephosphorylated GS, thereby relaying insulin stimulation to hepatic glycogen deposition. Importantly, this PP1-mediated signaling cascade is independent of GSK3. Therefore, we reveal a regulatory axis consisting of insulin/AKT/PPP1R3G/PPP1R3B that operates in parallel to the GSK3-dependent pathway, controlling glycogen synthesis and glucose homeostasis in insulin signaling. : Compelling evidence suggests that a GSK3-independent pathway exists, linking insulin signaling with glycogen synthesis. However, the molecular mechanism underlying this pathway remains unclear. Li et al. reveal the presence of an axis for insulin signaling consisting of insulin/AKT/PPP1R3G/PPP1R3B that controls glycogen synthesis and glucose homeostasis in parallel to the GSK3-dependent pathway.

NORFA, the first lincRNA associated with sow fertility, has been shown to control granulosa cell (GC) functions and follicular atresia. However, the underlying mechanism is not fully understood. In this study, RNA-seq was performed and we noticed that inhibition of NORFA led to dramatic transcriptomic alterations in porcine GCs. A total of 1,272 differentially expressed transcripts were identified, including 1167 DEmRNAs and 105 DEmiRNAs. Furthermore, protein–protein interaction, gene-pathway function, and TF–miRNA–mRNA regulatory networks were established and yielded four regulatory modules with multiple hub genes, such as AR, ATG5, BAK1, CENPE, NR5A1, NFIX, WNT5B, ssc-miR-27b, and ssc-miR-126. Functional assessment showed that these hub DEGs were mainly enriched in TGF-β, PI3K-Akt, FoxO, Wnt, MAPK, and ubiquitin pathways that are essential for GC states (apoptosis and proliferation) and functions (hormone secretion). In vitro, we also found that knockdown of NORFA in porcine GCs significantly induced cell apoptosis, impaired cell viability, and suppressed 17β-estradiol (E2) synthesis. Notably, four candidate genes for sow reproductive traits (INHBA, NCOA1, TGFβ-1, and TGFBR2) were also identified as potential targets of NORFA. These findings present a panoramic view of the transcriptome in NORFA-reduced GCs, highlighting that NORFA, a candidate lincRNA for sow fertility, is crucial for the normal states and functions of GCs.

Liver cancer is associated with high mortality, particularly in patients infected with the hepatitis B virus. Treatment methods remain very limited. Here, we explored the effects of 17β-estradiol (E2) on apoptosis of various liver cell lines (LO2, HepG2, and HepG2.2.15 cells). Within a certain concentration range, 17β-estradiol induced oxidative stress and apoptosis of HepG2 cells, downregulated ERα-36 expression, and increased Akt and Foxo3a phosphorylation. p-Foxo3a became localized around the nucleus but did not enter the organelle. The levels of mRNAs encoding manganese superoxide dismutase (MnSOD) and catalase, to the promoters of which Foxo3a binds to trigger gene expression, were significantly reduced in HepG2 cells. 17β-estradiol had no obvious effects on LO2 or HepG2.2.15 cells. We speculate that 17β-estradiol may induce oxidative stress in HepG2 cells by increasing Foxo3a phosphorylation, thus promoting apoptosis. This may serve as a new treatment for hepatocellular carcinoma.

TGF-β family signaling pathways, including TGF-β and BMP pathways, are widely involved in the regulation of health and diseases through downstream SMADs, which are also regulated by multiple validated mechanisms, such as genetic regulation, epigenetic regulation, and feedback regulation. However, it is still unclear whether R-SMADs or Co-SMAD can feedback regulate the TGF-β family signaling pathways in granulosa cells (GCs). In this study, we report a novel mechanism underlying the feedback regulation of TGF-β family signaling pathways, i.e., SMAD4, the only Co-SMAD, positive feedback activates the TGF-β family signaling pathways in GCs with a basal level of TGF-β ligands by interacting with the core promoters of its upstream receptors. Mechanistically, SMAD4 acts as a transcription factor, and feedback activates the transcription of its upstream receptors, including ACVR1B, BMPR2, and TGFBR2, of the canonical TGF-β signaling pathways by interacting with three coactivators (c-JUN, CREB1, and SP1), respectively. Notably, three different interaction modes between SMAD4 and coactivators were identified in SMAD4-mediated feedback regulation of upstream receptors through reciprocal ChIP assays. Our findings in the present study indicate for the first time that SMAD4 feedback activates the canonical TGF-β family signaling pathways in GCs, which improves and expands the regulatory mechanism, especially the feedback regulation modes of TGF-β family signaling pathways in ovarian GCs.

Adaptation to osmotic stress is crucial for bacterial growth and survival in changing environments. Although a large number of osmotic stress response genes have been identified in various bacterial species, how osmotic changes affect bacterial motility, biofilm formation, and colonization of host niches remains largely unknown. In this study, we report that the LrhA regulator is an osmoregulated transcription factor that directly binds to the promoters of the flhDC, eps, and opgGH operons and differentially regulates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production, synthesis of osmoregulated periplasmic glucans (OPGs), biofilm formation, and root colonization of the plant growth-promoting bacterium Pantoea alhagi LTYR-11Z. Further, we observed that the LrhA-regulated OPGs control RcsCD-RcsB activation in a concentration-dependent manner, and a high concentration of OPGs induced by increased medium osmolarity is maintained to achieve the high level of activation of the Rcs phosphorelay, which results in enhanced EPS synthesis and decreased motility in P. alhagi Moreover, we showed that the osmosensing regulator OmpR directly binds to the promoter of lrhA and promotes its expression, while lrhA expression is feedback inhibited by the activated Rcs phosphorelay system. Overall, our data support a model whereby P. alhagi senses environmental osmolarity changes through the EnvZ-OmpR two-component system and LrhA to regulate the synthesis of OPGs, EPS production, and flagellum-dependent motility, thereby employing a hierarchical signaling cascade to control the transition between a motile lifestyle and a biofilm lifestyle.IMPORTANCE Many motile bacterial populations form surface-attached biofilms in response to specific environmental cues, including osmotic stress in a range of natural and host-related systems. However, cross talk between bacterial osmosensing, swimming, and biofilm formation regulatory networks is not fully understood. Here, we report that the pleiotropic regulator LrhA in Pantoea alhagi is involved in the regulation of flagellar motility, biofilm formation, and host colonization and responds to osmotic upshift. We further show that this sensing relies on the EnvZ-OmpR two-component system that was known to detect changes in external osmotic stress. The EnvZ-OmpR-LrhA osmosensing signal transduction cascade is proposed to increase bacterial fitness under hyperosmotic conditions inside the host. Our work proposes a novel regulatory mechanism that links osmosensing and motile-sessile lifestyle transitions, which may provide new approaches to prevent or promote the formation of biofilms and host colonization in P. alhagi and other bacteria possessing a similar osmoregulatory mechanism.

Background and aims Hyperlipidemia-induced atherosclerosis is the major cause of heart attack and stroke in humans. However, pathological details and molecular mechanisms underlying early atherogenesis remain incompletely characterized. This study explored the early events of atherogenesis in a hypercholesterolemic zebrafish model in vivo. Methods We used transparent transgenic zebrafish larvae Tg(lysc:EGFP), Tg(mpx:EGFP), Tg(mpeg1:EGFP), Tg(flk1:EGFP) or Tg(lysc:EGFP/flk1:mCherry), together with fluorescently labeled control and high cholesterol diets (HCD), to dynamically investigate the early development of atherosclerosis with confocal in vivo. Endothelial cells with green fluorescence were sorted by fluorescence-activated cell sorting (FACS) to detect gene expression. Moreover, we treated hypercholesterolemic zebrafish model in vivo or human umbilical vein endothelial cells (HUVEC) in vitro with rosiglitazone, an agonist of peroxisome proliferator-activated receptor γ (PPARγ). Results We found that HCD-induced endothelial inflammation was an earlier pathological alteration than myeloid cells/neutrophils accumulation and lipid deposition in zebrafish vascular vessels of HCD-fed zebrafish. Endothelial inflammation was characterized by down-regulation of anti-inflammatory PPARγ and upregulation of pro-inflammatory tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β). Pharmacological treatment with rosiglitazone reversed the decrease in the expression of PPARγ and decreased expression of TNF-α and IL-1β in HCD-fed zebrafish. Moreover, rosiglitazone ameliorated myeloid cells accumulation and lipid deposition in HCD-fed zebrafish in vivo. Conclusions Hyperlipidemia-induced endothelial inflammation happens earlier than myeloid cell neutrophils accumulation in vascular vessels, and neutrophils accumulation is prior to lipid deposition during the initial stage of atherosclerosis. Early alleviation of inflammation induced by HCD would have a prophylactic effect for the initial development of atherosclerosis.

Aberrant loss of tumor-suppressor genes plays a crucial role in tumorigenesis and development of colorectal cancer (CRC). Extensive studies have reported tha hypermethylation of Ras association domain family member 6 (RASSF6) is common in various solid tumors. Another important mode of epigenetic regulation, microRNA (miRNA) regulation of RASSF6, is far from clear. The aim of the present work was to screen out novel miRNA regulating RASSF6, and to explore its underlying mechanism in CRC. With the use of bioinformatics, clinical sample data, and luciferase binding assay, we determined that microRNA-496 (miR-496) could be a novel oncomiR that directly binds to RASSF6. Next, a series of miR-496 mimics or inhibitor, or RASSF6 small interfering RNA (siRNA) introduced into CRC cells were applied to examine the effect of miR-496 on CRC cell viability, migration, and epithelial-mesenchymal transition (EMT). The results demonstrated that miR-496/RASSF6 could promote cell migration and EMT via Wnt signaling activation, but had no effect on cell viability. Our results confirmed that the miR-496/RASSF6 axis is involved in Wnt pathway-mediated tumor metastasis, highlighting its potential as a therapeutic target for CRC.

Both SMAD4 and miR-126* have been proven to be involved in granulosa cell (GC) apoptosis and even follicular atresia, through commonly regulating follicle-stimulating hormone receptor (FSHR), the FSH-specific transmembrane receptor of GCs. However, the regulatory relationship between them in GCs is still unknown. In this study, we report that SMAD4 suppresses the expression of miR-126* and impairs its function in GCs of the porcine ovary by acting as a transcription factor. A classic SMAD4-binding element (SBE) site was found in the promoter of miR-126* by using in silico methods. Luciferase assay, qRT-PCR, and ChIP assay proved that SMAD4 serves as a transcriptional repressor and directly binds to SBE site within miR-126* gene promoter, which further reduces miR-126* gene expression and inhibits its transcriptional activity in GCs. Furthermore, SMAD4 also controls miR-126*-mediated expression of FSHR (a direct target of miR-126* in GCs). In addition, we prove that SMAD4 induces CYP19A1 expression (encodes aromatase, the key enzyme for oestrogen biosynthesis) and inhibits GC apoptosis through the miR-126*/FSHR axis. Taken together, our findings not only established a direct link between SMAD4 and miRNA-126*, two key factors of GC apoptosis, but also revealed an important way in which the SMAD4 regulates GC function, the miRNA-126*/FSHR axis.

Oxidative stress (OS), a common intracellular phenomenon induced by excess reactive oxygen species (ROS) generation, has been shown to be associated with mammalian ovarian follicular development blockage and granulosa cell (GC) impairment. However, the mechanism involved in these effects remains unknown, and the effect of OS on the transcriptome profiles in porcine GCs has not been fully characterized. In this study, we found that hydrogen peroxide-mediated oxidative stress induced porcine GC apoptosis and impaired cell viability. Moreover, RNA-seq analysis showed that oxidative stress induced dramatic changes in gene expression in porcine GCs. A total of 2025 differentially expressed genes (DEGs) were identified, including 1940 DEmRNAs and 55 DEmiRNAs. Functional annotation showed that the DEGs were mainly associated with cell states and function regulation. In addition, multiple hub genes (FOXO1, SOD2, BMP2, DICER1, BCL2L11, FZD4, ssc-miR-424, and ssc-miR-27b) were identified by constructing protein-protein interaction and DEmiRNA-DEmRNA regulatory networks. Furthermore, a gene-pathway-function coregulatory network was established and demonstrated that these hub genes were enriched in FoxO, TGF-β, Wnt, PIK3-Akt, MAPK, and cAMP signaling pathways, which play important roles in regulating cell apoptosis, cell proliferation, stress responses, and hormone secretion. The current research provides a comprehensive perspective of the effects of oxidative stress on porcine GCs and also identifies potential therapeutic targets for oxidative stress-induced female infertility.

Summary Bioenergetic reprogramming during hypoxia adaption is critical to promote hepatocellular carcinoma (HCC) growth and progression. However, the mechanism underlying the orchestration of mitochondrial OXPHOS (oxidative phosphorylation) and glycolysis in hypoxia is not fully understood. Here, we report that mitochondrial UQCC3 (C11orf83) expression increases in hypoxia and correlates with the poor prognosis of HCC patients. Loss of UQCC3 impairs HCC cell proliferation in hypoxia in vitro and in vivo. Mechanistically, UQCC3 forms a positive feedback loop with mitochondrial reactive oxygen species (ROS) to sustain UQCC3 expression and ROS generation in hypoxic HCC cells and subsequently maintains mitochondrial structure and function and stabilizes HIF-1α expression to enhance glycolysis under hypoxia. Thus, UQCC3 plays an indispensable role for bioenergetic reprogramming of HCC cells during hypoxia adaption by simultaneously regulating OXPHOS and glycolysis. The positive feedback between UQCC3 and ROS indicates a self-modulating model within mitochondria that initiates the adaptation of HCC to hypoxic stress.

Canonical TGF-β signals are transduced from the cell surface to the cytoplasm, and then translocated into the nucleus, a process that involves ligands (TGF-β1), receptors (TGFBR2/1), receptor-activated SMADs (SMAD2/3), and the common SMAD (SMAD4). Here we provide evidence that SMAD4, a core component of the canonical TGF-β signaling pathway, regulates the canonical TGF-β signaling pathway in porcine granulosa cells (GCs) through a feedback mechanism. Genome-wide analysis and qRT-PCR revealed that SMAD4 affected miRNA biogenesis in GCs. Interestingly, TGFBR2, the type II receptor of the canonical TGF-β signaling pathway, was downregulated in SMAD4-silenced GCs and found to be a common target of SMAD4-inhibited miRNAs. miR-425, the most significantly elevated miRNA in SMAD4-silenced GCs, mediated the SMAD4 feedback regulation of the TGF-β signaling pathway. This was accomplished through a direct interaction between the transcription factor SMAD4 and the miR-425 promoter, and a direct interaction between miR-425 and the TGFBR2 3′-UTR. Furthermore, miR-425 enhanced GC apoptosis by targeting TGFBR2 and the canonical TGF-β signaling pathway, which was rescued by SMAD4 and TGF-β1. Overall, our findings demonstrate that a positive feedback mechanism exists within the canonical TGF-β signaling pathway. This study also provides new insights into mechanism underlying the canonical TGF-β signaling pathway, which regulates GC function and follicular development.

The cytochrome P450 family 19 subfamily A member 1 (CYP19A1) gene, encodes aromatase, a key enzyme in estradiol (E2) synthesis, and is down-regulated during porcine follicular atresia. However, its role in and the mechanism of transcriptional repression in follicular atresia is largely unknown. In the present study, we show that the CYP19A1 gene stimulates E2 release and inhibits cell apoptosis in porcine granulosa cells (GCs). SMAD4, an anti-apoptotic moderator, was identified as a transcription factor of the porcine CYP19A1 gene and enhanced the expression and function of CYP19A1 in porcine GCs through direct binding to a SMAD4-binding element (SBE) within the promoter region of CYP19A1 gene. Moreover, we found that miR-10b, a pro-apoptotic factor, directly interacted with 3'-UTR of the porcine CYP19A1 mRNA, inhibiting its expression and function in porcine GCs. Collectively, we demonstrated that CYP19A1 is an inhibitor of follicular atresia and is regulated by both SMAD4 and miR-10b. These findings provide further insight into the mechanisms of CYP19A1 in steroid hormone synthesis and GC apoptosis and provide molecular targets for exploring methods of treatment for steroid-dependent reproductive disorders.

miR-1275 is one of the microRNAs (miRNAs) that are differentially expressed during follicular atresia in pig ovaries, as identified by a miRNA microarray assay in our previous study [1]. However, its functions in follicular atresia remain unknown. In this study, we showed that miR-1275 promotes early apoptosis of porcine granulosa cells (pGCs) and the initiation of follicular atresia, and inhibits E2 release and expression of CYP19A1, the key gene in E2 production. Bioinformatics and luciferase reporter assays revealed that liver receptor homolog (LRH)-1, not CYP19A1, is a direct functional target of miR-1275. In vitro overexpression and knockdown experiments showed that LRH-1 significantly repressed apoptosis and induced E2 secretion and CYP19A1 expression in pGCs. LRH-1, whose expression was regulated by miR-1275, prevented apoptosis in pGCs. Furthermore, luciferase and chromatin immunoprecipitation assays demonstrated that LRH-1 protein bound to the CYP19A1 promoter and increased its activity. Our findings suggest that miR-1275 attenuates LRH-1 expression by directly binding to its 3'UTR. This prevents the interaction of LRH-1 protein with the CYP19A1 promoter, represses E2 synthesis, promotes pGC apoptosis, and initiates follicular atresia in porcine ovaries.

Mammalian ovarian follicular atresia is a complex and fine-regulated biological process with active involvement of connective tissue growth factor (CTGF). The emergence of studies of endogenous non-coding RNAs has raised a new aspect for exploration of the regulatory mechanisms involved in follicular atresia. Here, we aimed to illustrate a circRNA involved in the CTGF regulatory pathway during the apoptosis and follicular atresia of pig granulosa cells (GCs). We first detected a decreased expression pattern of CTGF during follicular atresia using IHC, FISH and qRT-PCR and confirmed the anti-apoptosis effect of CTGF in GCs in vitro by CTGF siRNA knockdown. Then, we used a dual luciferase activity assay to demonstrate CTGF as a direct functional target of miR-10a-5p, which was upregulated in atresic follicles and promoted the apoptosis of GCs in vitro. The negative effect of miR-10a-5p on GC viability was confirmed by cell cycle assays, cell proliferation/apoptosis assays and the WB detection of marker proteins. More importantly, we identified a novel circRNA, termed circINHA, that was downregulated during atresia in ovarian follicles, and we confirmed a direct interaction between miR-10a-5p and circINHA. Finally, we demonstrated that circINHA promoted GCs proliferation and inhibited GCs apoptosis via CTGF as a competing endogenous RNA (ceRNA) that directly bound to miR-10a-5p. Taken together, this study provides evidence for the circINHA/miR-10a-5p/CTGF regulatory pathway in follicular GC apoptosis and provides novel insights into the role of circRNAs in the modulation of ovarian physiological functions.

Androgen, which acts via the androgen receptor (AR), plays crucial roles in mammalian ovarian function. Recent studies showed that androgen/AR signaling regulates follicle-stimulating hormone receptor (FSHR) expression in follicles; however, the detailed mechanism underlying this regulation remained unknown. Here, we demonstrate that AR and miR-126* cooperate to inhibit FSHR expression and function in pig follicular granulosa cells (pGCs). In pGCs, overexpression of AR decreased, whereas knockdown increased,FSHRmRNA and protein expression; however, neither manipulation affectedFSHRpromoter activity. Using a dual-luciferase reporter assay, we found that theFSHRgene is a direct target of miR-126*, which inhibits FSHR expression and increases the rate of AR-induced apoptosis in pGCs. Collectively, our data show for the first time that the AR/miR-126* axis exerts synergetic effects in the regulation of FSHR expression and apoptosis in pGCs. Our findings thus define a novel pathway, AR/miR-126*/FSHR, that regulates mammalian GC functions.

During ovarian follicular growth and development, only a few follicles actually ovulate. Recently, it was found that follicular atresia is triggered by granulosa cell apoptosis, but the molecular mechanism of follicular atresia was not understood. Using flow cytometry, we found that miR-34a promotes granulosa cell apoptosis in pig ovarian follicles. In addition, inhibin beta B was found to be a miR- 34a target gene, based on luciferase reporter assays, quantitative RT- PCR and Western blotting. Taken together, our data indicate that miR- 34a plays an important role in granulosa cell apoptosis by targeting the INHBB gene in the porcine ovary.