Tricholoma matsutake polysaccharides suppress excessive melanogenesis via JNK-mediated pathway: Investigation in 8- methoxypsoralen induced B16-F10 melanoma cells and clinical study.

Skin hyperpigmentation is a worldwide condition associated with augmented melanogenesis. However, conventional therapies often entail various adverse effects. Here, we explore the safety range and depigmentary effects of polysaccharides extract of Tricholoma matsutake (PETM) in an in vitro model and further evaluated its efficacy at the clinical level. An induced-melanogenesis model was established by treating B16-F10 melanoma cells with 8-methoxypsoralen (8-MOP). Effects of PETM on cell viability and melanin content were examined and compared to a commonly used depigmentary agent, α-arbutin. Expressions of key melanogenic factors and upstream signaling pathway were analysed by quantitative PCR and western blot. Moreover, a placebo-controlled clinical study involving Chinese females with skin hyperpigmentation was conducted to measure the efficacy of PETM on improving facial pigmented spots, melanin index, and individual typology angle (ITA°). Results demonstrated that PETM (up to 0.5 mg/mL) had little effect on the viability and motility of B16-F10 cells. Notably, it significantly suppressed the melanin content and expressions of key melanogenic factors induced by 8-MOP in B16-F10 melanoma cells. Western blotting results revealed that PETM inhibited melanogenesis by inactivating c-Jun N-terminal kinase (JNK), and this inhibitory role could be rescued by JNK agonist treatment. Clinical findings showed that PETM treatment resulted in a significant reduction of facial hyperpigmented spot, decreased melanin index, and improved ITA° value compared to the placebo-control group. In conclusion, these in vitro and clinical evidence demonstrated the safety and depigmentary efficacy of PETM, a novel polysaccharide agent. The distinct mechanism of action of PETM on melanogenic signaling pathway positions it as a promising agent for developing alternative therapies.

Rhizoma Paridis saponins attenuate Gram-negative bacteria-induced inflammatory acne by binding to KEAP1 and modulating Nrf2 and MAPK pathways.

Acne vulgaris represents a chronic inflammatory condition, the pathogenesis of which is closely associated with the altered skin microbiome. Recent studies have implicated a profound role of Gram-negative bacteria in acne development, but there is a lack of antiacne agents targeting these bacteria. Polyphyllins are major components of Rhizoma Paridis with great anti-inflammatory potential. In this study, we aimed to evaluate the antiacne effects and the underlying mechanisms of PPH and a PPH-enriched Rhizoma Paridis extract (RPE) in treating the Gram-negative bacteria-induced acne. PPH and RPE treatments significantly suppressed the mRNA and protein expressions of interleukin (IL)-1ß and IL-6 in lipopolysaccharide (LPS)-induced RAW 264.7 and HaCaT cells, along with the intracellular reactive oxygen species (ROS) generation. Furthermore, PPH and RPE inhibited the nuclear translocation of nuclear factor kappa-B (NF-κB) P65 in LPS-induced RAW 264.7 cells. Based on molecular docking, PPH could bind to kelch-like ECH-associated protein 1 (KEAP1) protein. PPH and RPE treatments could activate nuclear factor erythroid 2-related factor 2 (NRF2) and upregulate haem oxygenase-1 (HO-1). Moreover, RPE suppressed the mitogen-activated protein kinase (MAPK) pathway. Therefore, PPH-enriched RPE showed anti-inflammatory and antioxidative effects in vitro, which is promising for alternative antiacne therapeutic.

Analyzing the cellular and molecular atlas of ovarian mesenchymal cells provides a strategy against female reproductive aging.

Ovarian mesenchymal cells (oMCs) constitute a distinct microenvironment that supports folliculogenesis under physiological conditions. Supplementation of exogenous non-ovarian mesenchymal-related cells has been reported to be an efficient approach to improve ovarian functions. However, the development and cellular and molecular characteristics of endogenous oMCs remain largely unexplored. In this study, we surveyed the single-cell transcriptomic landscape to dissect the cellular and molecular changes associated with the aging of oMCs in mice. Our results showed that the oMCs were composed of five ovarian differentiated MC (odMC) populations and one ovarian mesenchymal progenitor (oMP) cell population. These cells could differentiate into various odMCs via an oMP-derived route to construct the ovarian stroma structures. Comparative analysis revealed that ovarian aging was associated with decreased quantity of oMP cells and reduced quality of odMCs. Based on the findings of bioinformatics analysis, we designed different strategies involving supplementation with young oMCs to examine their effects on female fertility and health. Our functional investigations revealed that oMCs supplementation prior to ovarian senescence was the optimal method to improve female fertility and extend the reproductive lifespan of aged females in the long-term.

SRSF2 in Sertoli cells is essential for testicular development and spermatogenesis in mice.

Sertoli cells are essential for testis development and normal spermatogenesis by providing support and nutrients. Pre-messenger RNA (pre-mRNA) processing is the basic mechanism required for gene expression, and members of the serine/arginine-rich protein (SR) family are key components of the machines that perform these basic processing events. Serine/arginine-rich splicing factor 2 (SRSF2) is an important member of the SR family; however, the physiological functions of SRSF2 in Sertoli cells are still unclear. Here, we found that SRSF2 was localized in the nuclei of Sertoli and germ cells in male mice at all stages by breeding Amh-Cre mice obtained with Srsf2-specific knockout in Sertoli cells to define the function of SRSF2 in Sertoli cells. The experimental results showed that specific deletion of SRSF2 impaired fetal Sertoli cell proliferation and induced abnormal apoptosis and severe DNA damage in seminiferous tubules, resulting in severe testicular dysplasia, seminiferous tubule atrophy, and almost no normal seminiferous tubules at postnatal day 14. Eventually, these changes resulted in failure to produce normal sperm and infertility. Further RNA-seq results showed that many key genes related to proliferation and apoptosis were downregulated; Racgap1 mRNA undergoes exon skipping. Thus, SRSF2-dependent Sertoli cells are essential for testicular development and male reproduction.

SRSF1 regulates primordial follicle formation and number determination during meiotic prophase I.

BACKGROUND: Ovarian folliculogenesis is a tightly regulated process leading to the formation of functional oocytes and involving successive quality control mechanisms that monitor chromosomal DNA integrity and meiotic recombination. A number of factors and mechanisms have been suggested to be involved in folliculogenesis and associated with premature ovarian insufficiency, including abnormal alternative splicing (AS) of pre-mRNAs. Serine/arginine-rich splicing factor 1 (SRSF1; previously SF2/ASF) is a pivotal posttranscriptional regulator of gene expression in various biological processes. However, the physiological roles and mechanism of SRSF1 action in mouse early-stage oocytes remain elusive. Here, we show that SRSF1 is essential for primordial follicle formation and number determination during meiotic prophase I. RESULTS: The conditional knockout (cKO) of Srsf1 in mouse oocytes impairs primordial follicle formation and leads to primary ovarian insufficiency (POI). Oocyte-specific genes that regulate primordial follicle formation (e.g., Lhx8, Nobox, Sohlh1, Sohlh2, Figla, Kit, Jag1, and Rac1) are suppressed in newborn Stra8-GFPCre Srsf1Fl/Fl mouse ovaries. However, meiotic defects are the leading cause of abnormal primordial follicle formation. Immunofluorescence analyses suggest that failed synapsis and an inability to undergo recombination result in fewer homologous DNA crossovers (COs) in the Srsf1 cKO mouse ovaries. Moreover, SRSF1 directly binds and regulates the expression of the POI-related genes Six6os1 and Msh5 via AS to implement the meiotic prophase I program. CONCLUSIONS: Altogether, our data reveal the critical role of an SRSF1-mediated posttranscriptional regulatory mechanism in the mouse oocyte meiotic prophase I program, providing a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying primordial follicle formation.

Quantification of pharmacokinetic profiles of a recombinant canine PD-1 fusion protein by validated sandwich ELISA method.

Tumors are becoming a serious threat to the quality of life of human and dogs. Studies have shown that tumors have caused more than half of the deaths in older dogs. Similar to human, dogs will develop various and highly heterogeneous tumors, but there are currently no viable therapies for them. In human, immunotherapy has been used widely and considered as an effective treatment for tumors by immune checkpoint targets, which are also expressed on canine tumors, suggesting that immunotherapy may be a potential treatment for canine tumors. In this work, we developed a sandwich ELISA method to detect the concentration of recombinant canine PD-1 fusion protein in canine serum and investigated pharmacokinetics in canines after intravenous infusion administration. After being validated, the ELISA method showed an excellent linear relationship in 25.00-3,200.00 ng/ml in serum, and the R 2 was more than 0.99 with four-parameter fitting. The precision and accuracy of intra-assay and inter-assay at the five different concentrations met the requirements of quantitative analysis. At the same time, no hook effect was observed at the concentration above ULOQ, and the stability was good under different predicted conditions with accuracy > 80%. The pharmacokinetic study in dogs has shown that the recombinant canine PD-1 fusion protein exhibited a typical biphasic PK profile after intravenous infusion administration, and the linear pharmacokinetic properties were observed between 1.00 and 12.00 mg/kg. Meanwhile, the T1/2 after intravenous infusion administration with non-compartmental analysis was about 5.79 days.

Intracellular CircRNA imaging and signal amplification strategy based on the graphene oxide-DNA system.

CircRNA is a type of covalently closed circular RNA molecule that serves as a potential biomarker for the disease early diagnosis and clinical researches. To achieve living cell imaging of specific circRNA, we developed a novel graphene oxide (GO)-based catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) signal dual amplification system (GO-CHA-HCR, abbreviated GO-AR) for circ-Foxo3 imaging in living cells. The developed system consists of four types of designed hairpin DNA HP1, HP2, H1, and fluorophore-labeled H2, which are absorbed on the GO nanosheets surface leading to fluorescence quenching. In the presence of circ-Foxo3, the CHA cycle was initiated to form a hybrid chain with split fragments, which triggered the HCR cycle to generate dsDNA nanowires that were then released from GO. This process recovered the quenched fluorescence, realizing two-stage signal amplification. The GO-AR system effectively improved the signal-to-noise ratio compared to the traditional GO-CHA and GO-HCR detection system. The detection limit of circ-Foxo3 was as low as 15 pM with excellent sensitivity and selectivity. In addition, the enzyme-free sensing system was successfully applied in living cell circRNA imaging and serum circRNA detection, indicating its high potential in clinical diagnostics.

Amitriptyline plays important roles in modifying the ovarian morphology and improving its functions in rats with estradiol valerate-induced polycystic ovary.

Previous studies demonstrated that depression is more prevalent in women with polycystic ovary syndrome (PCOS). In this study, we aimed to determine whether amitriptyline (AMT), an antidepressant drug, plays a role in preventing PCOS. The results showed that AMT modified ovarian morphology improved the ovarian functions and estrus cycle in estradiol valerate (EV)-induced polycystic ovary (PCO). AMT restored the levels of estradiol (E2), testosterone (T) and progesterone (P4) to normal, and elevated the level of luteinizing hormone (LH) in EV-induced PCO. No significant changes in follicle stimulating hormone (FSH) levels were observed in rats with EV or AMT treatment. The restoration of norepinephrine (NE) level was detected in rats with EV-induced PCO. AMT also altered the expression levels of steroidogenesis genes and beta2-adrenoceptor (beta2-AR) in EV-induced PCO. Our data revealed that AMT improves the ovarian morphology and modifies ovarian expression of beta2-AR and steroidogenesis genes in rats with EV-induced rat PCO. Our data provide support for the hypothesis that AMT is considered as a candidate drug for preventing and treating PCOS along with depression.

Adipose tissues of MPC1± mice display altered lipid metabolism-related enzyme expression levels.

Mitochondrial pyruvate carrier 1 (MPC1) is a component of the MPC1/MPC2 heterodimer that facilitates the transport of pyruvate into mitochondria. Pyruvate plays a central role in carbohydrate, fatty, and amino acid catabolism. The present study examined epididymal white adipose tissue (eWAT) and intrascapular brown adipose tissue (iBAT) from MPC1± mice following 24 weeks of feeding, which indicated low energy accumulation as evidenced by low body and eWAT weight and adipocyte volume. To characterize molecular changes in energy metabolism, we analyzed the transcriptomes of the adipose tissues using RNA-Sequencing (RNA-Seq). The results showed that the fatty acid oxidation pathway was activated and several genes involved in this pathway were upregulated. Furthermore, qPCR and western blotting indicated that numerous genes and proteins that participate in lipolysis were also upregulated. Based on these findings, we propose that the energy deficiency caused by reduced MPC1 activity can be alleviated by activating the lipolytic pathway.

Mitigation of cell apoptosis induced by ochratoxin A (OTA) is possibly through organic cation transport 2 (OCT2) knockout.

Ochratoxin A (OTA) is a secondary metabolite of fungi such as Aspergillus ochraceus, A. niger and A. carbonarius, Penicillium verrucosum, and various other Penicillium, Petromyces, and Neopetromyces species. Various foods can be contaminated with OTA, potentially causing several toxic effects such as nephrotoxicity, hepatotoxicity and neurotoxicity. Typically, OTA is excreted by organic anion transporters (OATs). There is no research indicating organic cation transporters (OCTs) are involved in OTA nephrotoxicity. In our study, NRK-52E cells and rats were treated with OTA. OTA changed the expression of OCT1, OCT2 and OCT3 in NRK-52E cells and rat kidneys. TEA alleviated OTA-induced cell death, apoptosis, and DNA damage, and increased ROS. The OCT2 knockout cell line was constructed by the CRISPR/Cas 9 system. OCT2 knockout did not change the gene expression of OCT1, OAT1 and OAT3. OCT2 knockout alleviated the increase of Caspase 3 and CDK1 induced by OTA, leading to a reduction of apoptosis. In addition, OCT2 overexpression increased cell toxicity and expression of Caspase 3. In short, our findings indicate that OCT2 knockout possibly mitigate OTA-induced apoptosis by preventing the increase of Caspase 3 and CDK1.

Myricetin-induced brown adipose tissue activation prevents obesity and insulin resistance in db/db mice.

PURPOSE: Myricetin, a dietary flavonoid, is effective in the treatment of obesity and insulin resistance by increasing glucose transport and lipogenesis in adipocyte and diminishing systemic inflammation in obesity. However, it has not been revealed yet whether myricetin is associated with brown adipose tissue (BAT) activation that tightly mediates systemic energy metabolism. Therefore, this study assessed whether myricetin activated brown adipose tissue in db/db mouse. METHODS: Myricetin (400 mg/kg) in distilled water was fed daily by oral gavage to leptin receptor-deficient db/db male mice at 4 weeks of age for 14 weeks. Body weight change, glucose intolerance test, blood lipid profile and BAT activation using PET-CT were assessed. RESULTS: After myricetin treatment for 14 weeks, systemic insulin resistance and hepatic steatosis were significantly improved in db/db mice with body weight reduction and myricetin led to decreased adipocity, improved plasma lipid profiles and increased energy expenditure. Myricetin activated BAT by upregulating thermogenic protein expression and activating mitochondrial biogenesis, eventually increasing heat dissipation in skin after cold exposure. In iWAT, myricetin induced beige formation, increased thermogenic protein expression and activated mitochondrial biogenesis. Consistently, thermogenic gene expression was upregulated when myricetin was introduced in C3H10T1/2 cells during brown adipocytes differentiation. Moreover, the expression level of adiponectin was significantly increased in C3H10T1/2 cells, adipose tissues and plasma after myricetin treatment. CONCLUSIONS: These results highlight that myricetin prevents obesity and systemic insulin resistance by activating BAT and increasing adiponectin expression in BAT.

Limited Link between Oxidative Stress and Ochratoxin A-Induced Renal Injury in an Acute Toxicity Rat Model.

Ochratoxin A (OTA) displays nephrotoxicity and hepatotoxicity. However, in the acute toxicity rat model, there is no evidence on the relationship between OTA and nephrotoxicity and hepatotoxicity. Based on this, the integrated analysis of physiological status, damage biomarkers, oxidative stress, and DNA damage were performed. After OTA treatment, the body weight decreased and AST, ALP, TP, and BUN levels in serum increased. Hydropic degeneration, swelling, vacuolization, and partial drop occurred in proximal tubule epithelial cells. PCNA and Kim-1 were dose-dependently increased in the kidney, but Cox-2 expression and proliferation were not found in the liver. In OTA-treated kidneys, the mRNA expressions of Kim-1, Cox-2, Lcn2, and Clu were dose-dependently increased. The mRNA expressions of Vim and Cox-2 were decreased in OTA-treated livers. Some oxidative stress indicators were altered in the kidneys (ROS and SOD) and livers (SOD and GSH). DNA damage and oxidative DNA damage were not found. In conclusion, there is a limited link between oxidative stress and OTA-induced renal injury in an acute toxicity rat model.

miR-122 plays an important role in ochratoxin A-induced hepatocyte apoptosis in vitro and in vivo.

OTA can induce hepatotoxicity. Our previous research has shown that miRNAs play important roles in the OTA-induced hepatotoxicity. And miR-122 is the most abundant miRNA in the liver and is involved in diverse biological processes. This study was performed to clarify the role of miR-122 in OTA-induced hepatotoxicity. The expression levels of miR-122 and the target genes were quantified by real-time PCR. The OTA-induced apoptosis of hepatocyte and HepG2 cells was evaluated using a TUNEL kit, a CCK-8 kit, a flow cytometer and Hoechst 33342. miR-122 was inhibited in HepG2 cells. The results revealed that OTA affected rat hepatocyte apoptosis. miR-122 decreased at 4 weeks but increased at 13 weeks in the OTA-treated livers, and increased in the OTA-treated HepG2 cells; and the mRNA levels of CCNG1 and Bcl-w increased at 4 weeks and decreased at 13 weeks in the high-dose OTA-treatment groups and decreased in HepG2 cells. The apoptosis of HepG2 cells displayed a dose-related increase with OTA. However, the inhibition of miR-122 greatly reduced OTA-induced apoptosis. p53 decreased in vivo and in vitro. miR-122 is a primary effector of OTA-induced hepatocyte apoptosis through the CCNG1/p53 pathway and Bcl-w/caspase-3 pathway in vivo and in vitro. And miR-122 plays an important role in OTA-induced hepatotoxicity.

MiR-122 partly mediates the ochratoxin A-induced GC-2 cell apoptosis.

Ochratoxin A (OTA) is a mycotoxin which has been shown to be nephrotoxic, hepatotoxic, and immunotoxic to animals, and mainly exists in the mildew grain. MicroRNAs (miRNAs) regulate a wide variety of cellular processes. However, the toxic effects of OTA on the germ cell and whether miRNAs mediate the effects of OTA-induced GC-2 cell apoptosis are still not clear. In the present study, OTA treatment resulted in a dose-dependent increase apoptosis in GC-2 cells. MiR-122 was increased in the OTA-treated GC-2 cells. It showed that Bcl-w was down-regulated after OTA treatment, and caspase-3 was obviously activated. Cyclin G1 (CCNG1) was significantly decreased, and inversely the expression of p53 was increased. Inhibition of miR-122 partly relieved the OTA-induced GC-2 cell apoptosis. These results indicate that OTA induces GC-2 cell apoptosis by causing the increase of caspase-3 activity and that miR-122 partly mediates the OTA-induced cell apoptosis.

Mitochondrial proteomic analysis reveals the molecular mechanisms underlying reproductive toxicity of zearalenone in MLTC-1 cells.

Zearalenone (ZEA), a Fusarium mycotoxin that contaminates cereal crops worldwide, has been shown to affect the male reproductive system and trigger reactive oxygen species (ROS) generation. However, the mechanisms of its toxicity have not been fully understood. Because mitochondrion is a key organelle involved in producing ROS and generating metabolic intermediates for biosynthesis, an iTRAQ-based mitoproteomics approach was employed to identify the molecular mechanism of zearalenone toxicity using mitochondria of mouse Leydig tumor cells (MLTC-1). A total of 2014 nonredundant proteins were identified, among which 1401 proteins (69.56%) were overlapped. There were 52 differentially expressed proteins in response to ZEA, and they were primarily involved in energy metabolism, molecular transport and endocrine-related functions. Consistent with mitochondrial proteomic analysis, the ATP and intracellular Ca(2+) levels increased after ZEA treatment. The results suggest that lipid metabolism changed significantly after low-dose ZEA exposure, resulting in two alterations. One is the increase in energy production through promoted fatty acid uptake and ß-oxidation, along with excessive oxidative stress; the other is an inhibition of steroidogenesis and esterification, possibly resulting in reduced hormone secretion. A hypothetical model of ZEA-induced mitochondrial damage is proposed to provide a framework for the mechanism of ZEA toxicity.

Analysis of individual and combined effects of ochratoxin A and zearalenone on HepG2 and KK-1 cells with mathematical models.

Ochratoxin A (OTA) and Zearalenone (ZEA) are widespread mycotoxins that contaminate foodstuffs simultaneously, but sufficient data regarding their mixed toxicities are lacking. This study aims to analyze the style of combined effects of OTA and ZEA on cells of their target organs. For this purpose, cytotoxicity was determined in HepG2 and KK-1 cells treated with single and combined forms of OTA and ZEA. Furthermore, we have analyzed the data using two mathematical models based on the concepts of concentration addition (CA) and independent addition (IA). By analyzing data with nonlinear regression, toxins applied singly showed classic sigmoid dose-response curves in HepG2 cells whereas in KK-1 cells hormetic responses were observed. Exposure to equieffective mixtures of OTA and ZEA showed additive effects, irrespective of different nonlinear regression models used. Our results demonstrate that IA is an appropriate concept to account for mixture effects of OTA and ZEA. The results in ROS generation indicate a departure from additivity to antagonism or synergism at different concentrations, probably due to potential interaction during ROS production. This study shows that a risk assessment of mycotoxins should account for mixture effects, and prediction models are valuable tools for mixture assessment.

Prostaglandin F2α upregulates Slit/Robo expression in mouse corpus luteum during luteolysis.

Prostaglandin F2 α (PGF2 α) is a key factor in the triggering of the regression of the corpus luteum (CL). Furthermore, it has been reported that Slit/Robo signaling is involved in the regulation of luteolysis. However, the interactions between PGF2 α and Slit/Robo in the progression of luteolysis remain to be established. This study was designed to determine whether luteolysis is regulated by the interactions of PGF2 α and Slit/Robo in the mouse CL. Real-time PCR and immunohistochemistry results showed that Slit2 and its receptor Robo1 are highly and specifically co-expressed in the mouse CL. Functional studies showed that Slit/Robo participates in mouse luteolysis by enhancing cell apoptosis and upregulating caspase3 expression. Both in vitro and in vivo studies showed that PGF2 α significantly increases the expression of Slit2 and Robo1 during luteolysis through protein kinase C-dependent ERK1/2 and P38 MAPK signaling pathways, whereas an inhibitor of Slit/Robo signaling significantly decreases the stimulating effect of PGF2 α on luteolysis. These findings indicate that Slit/Robo signaling plays important roles in PGF2 α-induced luteolysis by mediating the PGF2 α signaling pathway in the CL.

LIM-homeodomain transcription factor Isl-1 mediates the effect of leptin on insulin secretion in mice.

In addition to the well known regulating effects of leptin on energy balance and glucose homeostasis through the central nervous system, circulating leptin has a direct effect on pancreatic islet and insulin secretion through its receptor (OBRb). The LIM-homeodomain transcription factor Isl-1 is expressed in all classes of pancreatic endocrine cells and is involved in regulating both islet development and insulin secretion. Both OBRb and Isl-1 mutations result in obesity-related diabetes. However, the interactions and physiological significance of leptin and Isl-1 in pancreatic islets remain to be established. Here, we show that most of leptin target cells in pancreatic islets and NIT beta cells express Isl-1. Both in vivo and in vitro results demonstrate that leptin suppresses Isl-1 expression and insulin secretion in islet in physiological and pathophysiological conditions, e.g. high fat diet. This effect of leptin on insulin secretion is lost in leptin receptor-defective db/db and Isl-1-inducible knock-out mice. We conclude that the action of leptin on insulin secretion is at least partly mediated by Isl-1. Another new finding of this study is that Isl-1 acts as a direct downstream target of leptin signaling molecule STAT3 to influence the effect of leptin on insulin secretion, whereas inversely, insulin has feedback regulating effects on Isl-1 expression through JAK-STAT3 pathway. These findings are crucial for understanding the mechanisms regulating insulin secretion and metabolism in related diseases, such as obesity and type 2 diabetes.

MicroRNA 375 mediates the signaling pathway of corticotropin-releasing factor (CRF) regulating pro-opiomelanocortin (POMC) expression by targeting mitogen-activated protein kinase 8.

Pro-opiomelanocortin (POMC) is a common precursor of melanocortin-related peptides in the pituitary and primarily regulated by corticotropin- releasing factor (CRF). Our results show that miR-375 is highly expressed in the mouse pituitary gland and located specifically in the intermediate lobe of pituitary. The functional studies show that the forced inhibition of endogenous miR-375 in AtT-20 mouse pituitary tumor cells and in the intermediate lobe of the pituitary gland significantly increases POMC expression, whereas miR-375 overexpression down-regulates POMC expression and ACTH secretion stimulated by CRF. This function of miR-375 is accomplished by its binding to the 3'-UTR of mitogen-activated protein kinase kinase kinase-8. Our results here have demonstrated that miR-375 acts as a negative regulating molecule mediating the signaling pathway of CRF and affecting POMC expression by targeting mitogen-activated protein kinase kinase kinase-8, which subsequently down-regulates ERK1/2 phosphorylation and nerve growth factor-induced clone B (NGFI-B) transcription activity. Taken together, our results show that miR-375 is a novel negative regulator of POMC expression and related hormone secretion.