MicroRNA-139-5p (miR-139-5p) is one of the most differentially expressed miRNAs in the brain between healthy people and depressed patients. However, its function in depression is unclear. Therefore, we investigated the function of miR-139-5p in depression. Here, miR-139-5p expression was found to be upregulated in the model group. MiR-139-5p inhibition could increase sucrose preference and decrease mice immobility time after chronic corticosterone (CORT) injection. Furthermore, compared with the antago-NC group, 3 weeks of antagomiR-139-5p treatment significantly decreased miR-139-5p level in model group hippocampus, increased sucrose preference index, reduced neuron damages, and enhanced the levels of nuclear receptor subfamily 3 group C member 1 (NR3C1), brain-derived neurotrophic factor (BDNF), phosphorylated/total tyrosine kinase receptor B (p-TrkB/TrkB), phosphorylated/total cAMP-response element-binding protein (p-CREB/CREB) and phosphorylated/total extracellular regulated protein kinases (p-ERK/ERK). Moreover, as a potential target for miR-139-5p, NR3C1 level was reduced by miR-139-5p mimic. Altogether, by activating the BDNF-TrkB signaling pathway, miR-139-5p inhibition plays an antidepressant-like role and might serve as an effective depression target (Fig. graphical abstract).

Nasopharyngeal carcinoma (NPC) is a subtype of head and neck cancer with dismal prognosis and high relapse rate. The role of long non-coding RNAs (lncRNAs) in NPC has become a research hotspot in recent years. This study aimed to interrogate the function and mechanism of lncRNA MSC antisense RNA 1 (MSC-AS1) in NPC.

Apoptosis is characterized by distinct morphological and biochemical changes that occur upon activation of a family of serine proteases known as caspases. Reactive oxygen species (ROS) induce apoptosis in many cell systems. Nuclear receptor subfamily 4, group A, member 1 (NR4A1) has been shown to induce apoptosis in a number of cell lineages, but can also paradoxically act as a death inhibitory factor. In the current study, we focused on the potential role of NR4A1 in hydrogen peroxide (H2O2)-induced apoptosis of normal human umbilical cord fibroblast (HUC-F2) cells.

BACKGROUND This study was designed to investigate the role of long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the proliferation as well as apoptosis of human umbilical vein endothelial cells (HUVECs), to offer a basis for therapy of hypertension. MATERIAL AND METHODS The lncRNA MALAT1 expression, hsa-miR-124-3p, hsa-miR-135a-5p, hsa-miR-135b-5p, and hsa-miR-455-5p in plasma were measured from 230 patients with hypertension and 230 non-hypertensive controls. The mechanism for lncRNA MALAT1 modulating the proliferation and apoptosis of HUVECs was explored by cell transfection, Cell Counting Kit-8 (CCK-8), quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase reporter assays. RESULTS The expression of hsa-miR-124-3p and hsa-miR-135a-5p was reduced and the expression of lncRNA MALAT1 was increased in the plasma of hypertensive patients. Moreover, the plasma levels of hsa-miR-124-3p and hsa-miR-135a-5p of hypertensive patients were negatively correlated with lncRNA MALAT1 (r=-0.64, -0.72; P<0.01, P<0.01, respectively). The level of nuclear receptor subfamily 3, group C, member 2 (NR3C2) protein was negatively correlated with hsa-miR-124-3p and hsa-miR-135a-5p (r=-0.74, -0.84; P<0.01, P<0.01, respectively). The proliferation of HUVECs was inhibited after the inhibition of MALAT. Additionally, after knocking down MALAT, the levels of hsa-miR-124-3p and hsa-miR-135a-5p in HUVECs were markedly increased, while the expression level of NR3C2 protein was decreased. The apoptotic rate of HUVECs after the transfection of MALAT1 small interfering RNA (si-MALAT1) (3.64±0.21%) was significantly reduced compared to that of transfected si-MALAT1 no template control (NC) (3.76±0.19%) and the control group (10.51±1.24%). CONCLUSIONS LncRNA MALAT1 regulates proliferation and apoptosis of HUVECs through the hsa-miR-124-3p/NR3C2 and/or hsa-miR-135a-5p/NR3C2 axis.

Hepatoblastoma is a kind of extreme malignancy frequently diagnosed in children. Although surgical resection is considered as the first-line treatment for hepatoblastoma, a relatively large population of patients have lost the preferred opportunity for surgery. Administration of locoregional ablation enables local tumor control but with the deficiency of insufficient ablation, residual tumor, and rapid progression. In this study, we integrated 219 hepatoblastoma and 121 non-cancer liver tissues to evaluate the expression of NR2F6, from which a higher NR2F6 level was found in hepatoblastoma compared with non-cancer livers with a standard mean difference (SMD) of 1.04 (95% CI: 0.79, 1.29). The overexpression of NR2F6 also appeared to be an efficient indicator in distinguishing hepatoblastoma tissues from non-cancer liver tissues from the indication of a summarized AUC of 0.90, with a pooled sensitivity of 0.76 and a pooled specificity of 0.89. Interestingly, nude mouse xenografts provided direct evidence that overexpressed NR2F6 was also detected in residual tumor compared to untreated hepatoblastoma. Chromatin immunoprecipitation-binding data in HepG2 cells and transcriptome analysis of HepG2 xenografts were combined to identify target genes regulated by NR2F6. We finally selected 150 novel target genes of NR2F6 in residual tumor of incomplete ablation, and these genes appeared to be associated with the biological regulation of lipid metabolism-related pathway. Accordingly, targeting NR2F6 holds a therapeutic promise in treating residual recurrent hepatoblastoma after incomplete ablation.

Cushing disease (CD) is a life-threatening disorder attributed to excess pituitary tumor-derived adrenocorticotrophic hormone (ACTH) and adrenal steroid secretion caused by pituitary tumors. Whereas CD was first described in 1932, the underlying genetic basis driving tumor growth and ACTH secretion remains unsolved. Here, we show that testicular orphan nuclear receptor 4 (TR4, nuclear receptor subfamily 2, group C, member 2) is overexpressed in human corticotroph tumors as well as in human and mouse corticotroph tumor cell lines. Forced overexpression of TR4 in both human and murine tumor cells increased proopiomelanocortin transcription, ACTH secretion, cellular proliferation, and tumor invasion rates in vitro. Conversely, knockdown of TR4 expression reversed all phenotypes. Mechanistically, we show that TR4 transcriptionally activates proopiomelanocortin through binding of a direct repeat 1 response element in the promoter, and that this is enhanced by MAPK-mediated TR4 phosphorylation. In vivo, TR4 overexpression promotes murine corticotroph tumor growth as well as enhances ACTH and corticosterone production, whereas TR4 knockdown decreases circulating ACTH and corticosterone levels in mice harboring ACTH-secreting tumors. Our findings directly link TR4 to the etiology of corticotroph tumors, hormone secretion, and cell growth as well as identify it as a potential target in the treatment of CD.

Nuclear receptor subfamily 2, group F, member 6 (NR2F6) is an orphan member of the nuclear receptor superfamily. Here, we show that genetic ablation of Nr2f6 significantly improves survival in the murine transgenic TRAMP prostate cancer model. Furthermore, Nr2f6(-/-) mice spontaneously reject implanted tumors and develop host-protective immunological memory against tumor rechallenge. This is paralleled by increased frequencies of both CD4(+) and CD8(+) T cells and higher expression levels of interleukin 2 and interferon γ at the tumor site. Mechanistically, CD4(+) and CD8(+) T cell-intrinsic NR2F6 acts as a direct repressor of the NFAT/AP-1 complex on both the interleukin 2 and the interferon γ cytokine promoters, attenuating their transcriptional thresholds. Adoptive transfer of Nr2f6-deficient T cells into tumor-bearing immunocompetent mice is sufficient to delay tumor outgrowth. Altogether, this defines NR2F6 as an intracellular immune checkpoint in effector T cells, governing the amplitude of anti-cancer immunity.

Nuclear receptor subfamily 4 group A member 2 (NR4A2) is an orphan nuclear receptor that is over-expressed in cancer and promotes cell proliferation, migration, transformation, and chemoresistance. Increased expression and function of NR4A2 have been attributed to various signaling pathways, but little is known about microRNA (miRNA) regulation of NR4A2 in cancer. To investigate the posttranscriptional regulation of NR4A2, we used a 3' untranslated region (UTR) reporter screen and identified miR-34 as a putative regulator of NR4A2. By using computer predictions, we identified and confirmed an miRNA recognition element in the 3' UTR of NR4A2 that was responsible for miR-34-mediated suppression. We next demonstrated that overexpression of exogenous miR-34 or activation of the p53 pathway, which regulates endogenous miR-34 expression, decreased NR4A2 expression. Consistent with previous reports, overexpression of NR4A2 blocked the induction of p53 target genes, including mir-34a. This was a phenotypic effect, as NR4A2 overexpression could rescue cells from p53-induced inhibition of proliferation. In summary, our results are the first characterization of a cancer-related miRNA capable of regulating NR4A2 and suggest a network and possible feedback mechanism involving p53, miR-34, and NR4A2.

The present study evaluated expression patterns of chemokine (C-C motif) ligand 2 gene/Monocyte chemoattractant protein-1 gene (CCL2/MCP-1), prostaglandin F2 alpha receptor gene (PTGFR) and immediate early genes including nuclear receptor subfamily 4, group A, member 1 (NR4A1), early growth response 1 (EGR1) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in cells of the bovine corpus luteum after intrauterine infusion of a low dose of prostaglandin F2α (PGF2A) aimed at enhancing our understanding of the mechanisms of luteolysis. Holstein dairy cows were superovulated (>6 corpora lutea [CL]) and on day 9 of the estrous cycle were infused with a low dose of PGF2A (0.5 mg PGF2A in 0.25 ml phosphate buffered saline) into the greater curvature of the uterine horn ipsilateral to the CL. Ultrasound-guided biopsy samples of different CL were collected at 0 min, 15 min, 30 min, 1h, 2h and 6h after PGF2A infusion. Expression profiles and localization of mRNA for PTGFR, CCL2/MCP-1, and immediate early genes (NR4A1, EGR1 and FOS), were investigated by using qPCR and in situ hybridization. The concentrations of early response genes including FOS, NR4A1, and EGR1 exhibited the greatest increase at 30 min after PGF2A, compared to other time points. Expression profile of CCL2 mRNA increased gradually after intrauterine infusion of PGF2A with maximal up-regulation for CCL2 at 6h. Abundance of PTGFR mRNA only increased at 15 min and significantly decreased at 6h, compared to 0 min. Cellular localizations of all studied genes except CCL2 (primarily localized to apparent immune cells) were predominantly visualized in large luteal cells. Interestingly, early response genes demonstrated a changing profile in cellular localization with initial responses appearing to be in both large luteal cells and endothelial cells, although no staining for PTGFR mRNA was observed in endothelial cells. Later, sustained responses, were only observed in large luteal cells, although PTGFR mRNA was decreasing in large luteal cells over time after PGF2A. The involvement of the immune system was also highlighted by the immediate increases in CCL2 mRNA that became much greater over time as there was an apparent influx of CCL2-positive immune cells. Thus, the temporal and cell-specific localization patterns for the studied mRNA demonstrate the complex pathways that are responsible for initiation of luteolysis in the bovine CL.

Obesity is a severe disease worldwide. Mitochondrial autophagy (mitophagy) may be related to metabolic abnormalities in obese individuals, but the mechanism is still unclear. We aimed to investigate whether nuclear receptors NR1D1 and ULK1 influence obesity by affecting mitophagy. In vitro model was established by inducing 3T3-L1 cells differentiation. MTT was detected cell viability. ELISA was tested triglyceride (TG). Oil red O staining was performed to detect lipid droplets. Flow cytometry was measured mtROS. ChIP and Dual-luciferase reporter assay were verified NR1D1 bind to ULK1. LC3 level was detected by IF. After differentiation medium treatment, cell viability was decreased, TG content and lipid droplets were increased Moreover, NR1D1 expression was reduced in Model group. NR1D1 overexpression was increased cell viability, reduced TG content and lipid droplets. Subsequently, NR1D1 inhibited TOM20 and mtROS, whereas, Parkin and PINK1 were accelerated. NR1D1 overexpression facilitated LC3 expression, whereas ULK1 knockdown was reversed the effect of NR1D1 overexpression. Liensinine also reversed the effect of NR1D1 overexpression, that is, cell viability was reduced, mtROS, TG content and lipid droplets were increased. The combination of nuclear receptor NR1D1 and ULK1 promoted mitophagy in adipocytes to alleviate obesity, which provided new target and strategy for obesity treatment.Abbreviations: Mitochondrial autophagy (mitophagy), triglyceride (TG), Uncoordinated-51 like autophagy activating kinase 1 (ULK1), Nuclear receptor subfamily 1 group D member 1 (NR1D1), American Type Culture Collection (ATCC), fetal bovine serum (FBS), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), short hairpin RNA ULK1 (sh-ULK1), wild-type (WT), mutant (MUT), Enzyme-linked immunosorbent assay (ELISA), mitochondrial reactive oxygen species (mtROS), Chromatin immunoprecipitation (ChIP), Quantitative real-time PCR (qRT-PCR), Immunofluorescence (IF), standard deviation (SD).

In males, Leydig cells are the main producers of testosterone and insulin-like 3 (INSL3), two hormones essential for sex differentiation and reproductive functions. Chicken ovalbumin upstream promoter-transcription factors I (COUP-TFI/NR2F1) and COUP-TFII (NR2F2) belong to the steroid/thyroid hormone nuclear receptor superfamily of transcription factors. In the testis, COUP-TFII is expressed and plays a role in the differentiation of cells committed to give rise to fully functional steroidogenic adult Leydig cells. Steroid production has also been shown to be diminished in COUP-TFII-depleted Leydig cells, indicating an important functional role in steroidogenesis. Until now, only a handful of target genes have been identified for COUP-TFII in Leydig cells. To provide new information into the mechanism of action of COUP-TFII in Leydig cells, we performed microarray analyses of COUP-TFII-depleted MA-10 Leydig cells. We identified 262 differentially expressed genes in COUP-TFII-depleted MA-10 cells. Many of the differentially expressed genes are known to be involved in lipid biosynthesis, lipid metabolism, male gonad development, and steroidogenesis. We validated the microarray data for a subset of the modulated genes by RT-qPCR. Downregulated genes included hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1), cytochrome P450, family 11, subfamily a, polypeptide 1 (Cyp11a1), prolactin receptor (Prlr), nuclear receptor subfamily 0, group B, member 2 (Shp/Nr0b2), ferredoxin 1 (Fdx1), scavenger receptor class B, member 1 (Scarb1), inhibin alpha (Inha), and glutathione S-transferase, alpha 3 (Gsta3). Finally, analysis of the Gsta3 and Inha gene promoters showed that at least two of the downregulated genes are potentially new direct targets for COUP-TFII. These data provide new evidence that further strengthens the important nature of COUP-TFII in steroidogenesis, androgen homeostasis, cellular defense, and differentiation in mouse Leydig cells.

The role, magnitude, and molecular nature of trans-driven expression variation underlying the upregulation of detoxification genes in pesticide resistant arthropod populations has remained enigmatic. In this study, we performed expression quantitative trait locus (eQTL) mapping (n = 458) between a pesticide resistant and a susceptible strain of the generalist herbivore and crop pest Tetranychus urticae. We found that a single trans eQTL hotspot controlled large differences in the expression of a subset of genes in different detoxification gene families, as well as other genes associated with host plant use. As established by additional genetic approaches including RNAi gene knockdown, a duplicated gene with a nuclear hormone receptor HR96-related ligand-binding domain was identified as causal for the expression differences between strains. The presence of a large family of HR96-related genes in T. urticae may enable modular control of detoxification and host plant use genes, facilitating this species' known and rapid evolution to diverse pesticides and host plants.

Glucocorticoids are widely used to treat inflammatory disorders; however, prolonged use of glucocorticoids results in side effects including osteoporosis, diabetes and obesity. Compound A (CpdA), identified as a selective NR3C1/glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1) modulator, exhibits an inflammation-suppressive effect, largely in the absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated in bone marrow-derived macrophages using an unbiased proteomics approach. We found that the autophagy receptor SQSTM1 but not NR3C1 mediates the anti-inflammatory action of CpdA. CpdA drives SQSTM1 upregulation by recruiting the NFE2L2 transcription factor to its promoter. In contrast, the classic NR3C1 ligand dexamethasone recruits NR3C1 to the Sqstm1 promoter and other NFE2L2-controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA induce autophagy, with marked different autophagy characteristics and morphology. Suppression of LPS-induced Il6 and Ccl2 genes by CpdA in macrophages is hampered upon Sqstm1 silencing, confirming that SQSTM1 is essential for the anti-inflammatory capacity of CpdA, at least in this cell type. Together, these results demonstrate how off-target mechanisms of selective NR3C1 ligands may contribute to a more efficient anti-inflammatory therapy.

Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is a nuclear receptor involved in gonadal and adrenal development. We identified a novel C-terminally truncating NR5A1 mutation, p.Leu423Trpfs*7, in dizygotic twins with 46,XY disorders of sex development. Our results highlight the functional importance of C-terminal region of NR5A1 and indicate that NR5A1 mutations can be associated with intrafamilial phenotypic variations, progressive testicular dysfunction, hypogonadotropic hypogonadism, and borderline adrenal dysfunction.

The AMP-activated protein kinase alpha (AMPKα) pathway has widely been considered a key factor in energy metabolism. Ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl] acetate (TMPA) is a novel AMPK agonist, which influences the stability of Nuclear Receptor Subfamily 4, Group A, Member 1 (Nur77)-serine-threonine kinase 11 (LKB1) in the nucleus. A recent study has determined that TMPA can ameliorate the reduction of insulin resistance in type II db/db mice. However, the role of TMPA in hepatocyte lipid metabolism has not been elucidated.

Indoxyl sulfate (IS) is a chronic kidney disease (CKD)-specific renal osteodystrophy metabolite that affects the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a transcription factor promoting osteoclastogenesis. However, the mechanisms underlying the regulation of NFATc1 by IS remain unknown. It is intriguing that the Aryl hydrocarbon receptor (AhR) plays a key role in osteoclastogenesis, since IS is an endogenous AhR agonist. This study investigates the relationship between IS concentration and osteoclast differentiation in Raw 264.7 cells, and examines the effects of different IS concentrations on NFATc1 expression through AhR signaling. Our data suggest that both osteoclastogenesis and NFATc1 are affected by IS through AhR signaling in both dose- and time-dependent manners. Osteoclast differentiation increases with short-term, low-dose IS exposure and decreases with long-term, high-dose IS exposure. Different IS levels switch the role of AhR from that of a ligand-activated transcription factor to that of an E3 ubiquitin ligase. We found that the AhR nuclear translocator may play an important role in the regulation of these dual functions of AhR under IS treatment. Altogether, this study demonstrates that the IS/AhR/NFATc1 signaling axis plays a critical role in osteoclastogenesis, indicating a potential role of AhR in the pathology and abnormality of bone turnover in CKD patients.

Understanding complex epigenetic mechanisms is necessary to fully elucidate the effects of antipsychotic drug. This study investigated DNA methylation and mRNA expression levels of dopamine D2 and D1 receptor (Drd2 and Drd1, respectively), nuclear receptor subfamily 3, group C, member 1 (Nr3c1) and stathmin 1 (Stmn1) in brain regions of mice exposed to social defeat stress (SDS) and effects of risperidone on altered methylation and mRNA expression levels induced by SDS.

Lymphatic endothelial cells arise from the venous endothelial cells in embryonic lymphatic development. However, the molecular mechanisms remain to be elucidated. We here report that prostaglandin (PG) E2 plays essential roles in the embryonic lymphatic development through the EP3 receptor, one of the PGE2 receptors. Knockdown of the EP3 receptor or inhibition of cyclooxygenases (COX; rate-limiting enzymes for PG synthesis) impaired lymphatic development by perturbing lymphatic specification during zebrafish development. These impairments by COX inhibition were recovered by treatment with sulprostone (EP1/3 agonist). Knockdown of the EP3 receptor further demonstrated its requirement in the expression of sex determining region Y-box 18 (sox18) and nuclear receptor subfamily 2, group F, member 2 (nr2f2), essential factors of the lymphatic specification. The EP3 receptor was expressed in the posterior cardinal vein (region of embryonic lymphatic development) and the adjacent intermediate cell mass (ICM) during the lymphatic specification. COX1 was expressed in the region more upstream of the posterior cardinal vein relative to the EP3 receptor, and the COX1-selective inhibitor impaired the lymphatic specification. On the other hand, two COX2 subtypes did not show distinct sites of expression around the region of expression of the EP3 receptor. Finally, we generated EP3-deficient zebrafish, which also showed defect in lymphatic specification and development. Thus, we demonstrated that COX1-derived PGE2-EP3 pathway is required for embryonic lymphatic development by upregulating the expression of key factors for the lymphatic specification.

Oxidative stress activates macroautophagy/autophagy and contributes to atherogenesis via lipophagic flux, a form of lipid removal by autophagy. However, it is not known exactly how endogenous antioxidant enzymes are involved in lipophagic flux. Here, we demonstrate that the antioxidant PRDX1 (peroxiredoxin 1) has a crucial role in the maintenance of lipophagic flux in macrophages. PRDX1 is more highly expressed than other antioxidant enzymes in monocytes and macrophages. We determined that Prdx1 deficiency induced excessive oxidative stress and impaired maintenance of autophagic flux in macrophages. Prdx1-deficient macrophages had higher intracellular cholesterol mass and lower cholesterol efflux compared with wild type. This perturbation in cholesterol homeostasis was due to impaired lipophagic cholesterol hydrolysis caused by excessive oxidative stress, resulting in the inhibition of free cholesterol formation and the reduction of NR1H3 (nuclear receptor subfamily 1, group H, member 3) activity. Notably, impairment of both lipophagic flux and cholesterol efflux was restored by the 2-Cys PRDX-mimics ebselen and gliotoxin. Consistent with this observation, apoe -/- mice transplanted with bone marrow from prdx1-/-apoe-/- mice had increased plaque formation compared with apoe-/- BM-transplanted recipients. This study reveals that PRDX1 is crucial to regulating lipophagic flux and maintaining macrophage cholesterol homeostasis against oxidative stress. We suggest that PRDX1-dependent control of oxidative stress may provide a strategy for treating atherosclerosis and autophagy-related human diseases.

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase β-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.