Quantitative transcriptomic and proteomic analysis reveals corosolic acid inhibiting bladder cancer via suppressing cell cycle and inducing mitophagy in vitro and in vivo.

Corosolic acid (CA) is a plant-derived terpenoid compound with many health benefits. However, the anti-tumor effects of CA in bladder cancer remain unexplored. Here, we found that CA inhibited bladder tumor both in vitro and in vivo, and had no significant toxicity in mice. With the aid of transcriptomics and proteomics, we elucidated the regulatory network mechanism of CA inhibiting bladder cancer. Through cell viability detection, cell fluorescence staining and flow cytometry, we discovered that CA inhibited bladder cancer mainly through blocking cell cycle. Interestingly, CA played anticancer roles by distinct mechanisms at different concentrations: low concentrations (<7.0 µg/ml) of CA mainly inhibited DNA synthesis by downregulating TOP2A and LIG1, and diminished mitosis by downregulating CCNA2, CCNB1, CDC20, and RRM2; high concentrations (≥7.0 µg/ml) of CA induced cell death through triggering mitophagy via upregulating NBR1, TAXBP1, SQSTM1/P62, and UBB. CA, as a natural molecule of homology of medicine and food, is of great significance for the prevention and treatment of cancer patients following clarifying its anti-cancer mechanism. This study provides a comprehensive understanding of the pharmacological mechanism of CA inhibition in bladder cancer, which is helpful for the development of new anti-tumor drugs based on CA.

The hepatic FOXQ1 transcription factor regulates glucose metabolism in mice.

AIM/HYPOTHESIS: Hepatic forkhead box q1 (FOXQ1) expression levels are regulated by nutritional and pathophysiological status. In this study we investigated the role of FOXQ1 in the regulation of hepatic gluconeogenesis. METHODS: We used multiple mouse and cell models to study the role of FOXQ1 in regulating expression of gluconeogenic genes, and cellular and hepatic glucose production. RESULTS: Expression of hepatic FOXQ1 was regulated by fasting in normal mice and was dysregulated in diabetic mice. Overexpression of FOXQ1 in primary hepatocytes inhibited expression of gluconeogenic genes and decreased cellular glucose output. Hepatic FOXQ1 rescue in db/db and high-fat diet-induced obese mice markedly decreased blood glucose level and improved glucose intolerance. In contrast, wild-type C57 mice with hepatic FOXQ1 deficiency displayed increased blood glucose levels and impaired glucose tolerance. Interestingly, studies into molecular mechanisms indicated that FOXQ1 interacts with FOXO1, thereby blocking FOXO1 activity on hepatic gluconeogenesis, preventing it from directly binding to insulin response elements mapped in the promoter region of gluconeogenic genes. CONCLUSIONS/INTERPRETATION: FOXQ1 is a novel factor involved in regulating hepatic gluconeogenesis, and the decreased FOXQ1 expression in liver may contribute to the development of type 2 diabetes.

Forkhead Box P1 (FOXP1) Transcription Factor Regulates Hepatic Glucose Homeostasis.

Dysregulation of hepatic gluconeogenesis contributes to the pathogenesis of diabetes, yet the detailed molecular mechanisms remain to be fully elucidated. Here we show that FOXP1, a transcriptional repressor, plays a key role in the regulation of systemic glucose homeostasis. Hepatic expression levels of FOXP1 are decreased in diabetic mice. Modest hepatic overexpression of FOXP1 in mice inhibited the expression of gluconeogenic genes, such as peroxisome proliferators-activated receptor γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6PC), leading to a decrease in hepatic glucose production and fasting blood glucose levels in normal mice and different mouse models of diabetes, including db/db diabetic and high-fat diet-induced obese mice. FOXP1 physically interacted with FOXO1 in vivo and competed with FOXO1 for binding to the insulin response element in the promoter region of gluconeogenic genes, thereby interfering expression of these genes. These results identify a previously unrecognized role for FOXP1 in the transcriptional control of hepatic glucose homeostasis.

Hepatic differentiated embryo-chondrocyte-expressed gene 1 (Dec1) inhibits sterol regulatory element-binding protein-1c (Srebp-1c) expression and alleviates fatty liver phenotype.

Hepatic steatosis, characterized by ectopic hepatic triglyceride accumulation, is considered as the early manifestation of non-alcoholic fatty liver diseases (NAFLD). Increased SREBP-1c level and activity contribute to excessive hepatic triglyceride accumulation in NAFLD patients; however, negative regulators of Srebp-1c are not well defined. In this study, we show that Dec1, a critical regulator of circadian rhythm, negatively regulates hepatic Srebp-1c expression. Hepatic Dec1 expression levels are markedly decreased in NAFLD mouse models. Restored Dec1 gene expression levels in NAFLD mouse livers decreased the expression of Srebp-1c and lipogenic genes, subsequently ameliorating the fatty liver phenotype. Conversely, knockdown of Dec1 expression by an adenovirus expressing Dec1-specific shRNA led to an increase in hepatic TG content in normal mouse livers. Correspondingly, expression levels of lipogenic genes, including Srebp-1c, Fas, and Acc, were increased in livers of mice with Dec1 knockdown. Moreover, a functional lipogenesis assay suggested that Dec1 overexpression repressed lipid synthesis in primary hepatocytes. Finally, a luciferase reporter gene assay indicates that DEC1 inhibits Srebp-1c gene transcription via the E-box mapped to the promoter region. Chromatin immunoprecipitation confirmed that DEC1 proteins bound to the identified E-box element. Our studies indicate that DEC1 is an important regulator of Srebp-1c expression and links circadian rhythm to hepatic lipogenesis. Activation of Dec1 can alleviate the nonalcoholic fatty liver phenotype.

MicroRNA-29a-c decrease fasting blood glucose levels by negatively regulating hepatic gluconeogenesis.

BACKGROUND & AIMS: The expression levels of microRNA-29 (miR-29) family members (miR-29a, miR-29b, miR-29c, here denoted collectively as miR-29a-c) are increased in livers of Goto-Kakizaki diabetic rats and db/db diabetic mice. However, the functional consequences of miR-29a-c upregulation in diabetic livers are not explored. The objective of this study was to evaluate the roles of miR-29a-c in the regulation of hepatic glucose production and blood glucose levels using different mouse models. METHODS: db/m, db/db diabetic and diet-induced obese (DIO) mice were injected with adenovirus expressing miR-29a-c through the tail vein. Blood glucose levels were measured and glucose-tolerance tests and pyruvate-tolerance tests were performed. To explore the molecular mechanism by which miR-29a-c regulate hepatic glucose metabolism, gain or loss of miR-29a-c function studies were performed in primary mouse hepatocytes and the direct effectors of miR-29-mediated effects on glucose metabolism were identified. RESULTS: Adenovirus-mediated overexpression of miR-29a-c in the livers of db/m, db/db, and DIO mice decreased fasting blood glucose levels and improved glucose tolerance. Overexpression of miR-29a-c in primary hepatocytes and mouse livers decreased the protein levels of PGC-1α and G6Pase, the direct targets of miR-29a-c, thereby reducing cellular, and hepatic glucose production. In contrast, loss of miR-29a-c function in primary hepatocytes increased the protein levels of PGC-1α and G6Pase and increased cellular glucose production. Finally, enforced expression of PGC-1α increased miR-29a-c expression levels in primary hepatocytes, thus forming a negative feedback regulation loop. CONCLUSIONS: miR-29a-c can regulate hepatic glucose production and glucose tolerance in mice.

Mouse KLF11 regulates hepatic lipid metabolism.

BACKGROUND & AIMS: Missense mutations in human Krüppel-like factor 11 (KLF11) lead to the development of diabetes, as a result of impaired insulin synthesis in the pancreas. However, the role of KLF11 in peripheral tissues is largely unknown. The aim of this study is to evaluate the role of KLF11 in the regulation of hepatic lipid homeostasis using different mouse models. METHODS: Adenoviruses expressing KLF11 (Ad-KLF11) or KLF11-specific shRNA (Ad-shKLF11) were injected into db/db diabetic, high-fat diet-induced obese (DIO), or normal C57BL/6J mice. Histological analysis of the fatty liver phenotype and biochemical analysis of hepatic and serum TG levels in these mice were performed. The molecular mechanism by which KLF11 regulates lipid metabolism in primary hepatocytes and mouse livers was explored. RESULTS: The expression of the transcription factor KLF11 gene is dysregulated in the livers of db/db and DIO mice. Adenovirus-mediated overexpression of KLF11 in the livers of db/db and DIO mice activates the PPARα signaling pathway, subsequently markedly improving the fatty liver phenotype. Conversely, knockdown of KLF11, by adenovirus (Ad-shKLF11) in livers of wild type C57BL/6J and db/m mice, increases hepatic triglyceride (TG) levels, owing to decreased fatty acid oxidation. Finally, the treatment of diabetic mice with Ad-shPPARα abolishes KLF11 stimulatory effects on the expression of genes involved in fatty acid oxidation and inhibitory effects on hepatic TG content. In contrast, PPARα rescue restores the increased hepatic TG levels in Ad-shKLF11-infected db/m mice to normal levels. CONCLUSIONS: KLF11 is an important regulator of hepatic lipid metabolism.

Mouse patatin-like phospholipase domain-containing 3 influences systemic lipid and glucose homeostasis.

UNLABELLED: Human patatin-like phospholipase domain-containing 3 (PNPLA3) is associated with increased liver fat content and liver injury. Here, we show that nutritional status regulates PNPLA3 gene expression in the mouse liver. Sterol response element binding protein-1 (SREBP-1) activated PNPLA3 gene transcription via sterol regulatory elements (SREs) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that SREBP-1 proteins bound to the identified SREs. Furthermore, SREBP-1c mediated the insulin and liver X receptor agonist TO901317-dependent induction of PNPLA3 gene expression in hepatocytes. Adenovirus-mediated overexpression of mouse PNPLA3 increased intracellular triglyceride content in primary hepatocytes, and knockdown of PNPLA3 suppressed the ability of SREBP-1c to stimulate lipid accumulation in hepatocytes. Finally, the overexpression of PNPLA3 in mouse liver increased the serum triglyceride level and impaired glucose tolerance; in contrast, the knockdown of PNPLA3 in db/db mouse liver improved glucose tolerance. CONCLUSION: Our data suggest that mouse PNPLA3, which is a lipogenic gene directly targeted by SREBP-1, promotes lipogenesis in primary hepatocytes and influences systemic lipid and glucose metabolism.

Sterol-regulatory-element-binding protein 1c mediates the effect of insulin on the expression of Cidea in mouse hepatocytes.

Members of the Cide [cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector] gene family have been reported to be associated with lipid metabolism. In the present study, we show that Cidea mRNA levels are markedly reduced by fasting and are restored upon refeeding in mouse livers. To elucidate the molecular mechanism, the promoter region of the mouse Cidea gene was analysed and a putative SRE (sterol-regulatory element) was identified. Studies using luciferase reporter constructs together with electrophoretic mobility-shift assays and chromatin immunoprecipitation confirmed the binding of SREBP-1c (SRE-binding protein 1c) to the putative SRE. Furthermore, adenovirus-mediated overexpression of SREBP-1c led to a dramatic increase in Cidea mRNA. In contrast with the induction of Cidea expression by insulin and TO901317 in wild-type mouse hepatocytes, the stimulatory effects were lost in hepatocytes prepared from SREBP-1c-null mice. Adenovirus-mediated overexpression of Cidea in hepatocytes promoted lipid accumulation and triacylglycerol (triglyceride) storage; however, knockdown of Cidea compromised the ability of SREBP-1c to stimulate lipid accumulation. Taken together, these results suggest that SREBP-1c directly mediates the effect of insulin on Cidea in hepatocytes and that Cidea, at least in part, mediates SREBP-1c-dependent lipid accumulation.

PGC-1alpha coactivates estrogen-related receptor-alpha to induce the expression of glucokinase.

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) is a key regulator of cellular energy metabolism and regulates processes such as adaptive thermogenesis, hepatic gluconeogenesis, fatty acid oxidation, and mitochondrial biogenesis by coactivating numerous nuclear receptors and transcription factors. Here, we demonstrate the presence of the ERRalpha binding site in the regulatory sequence of the glucokinase gene and that PGC-1alpha coactivates ERRalpha to stimulate the transcription of glucokinase. Simultaneous overexpression of PGC-1alpha and ERRalpha potently induced the glucokinase gene expression and its enzymatic activity in primary hepatocytes; however, expression of either PGC-1alpha or ERRalpha alone had no significant effect. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed the interaction of ERRalpha with the glucokinase promoter. Finally, the knockdown of endogenous ERRalpha with specific siRNA (siERRalpha) or pharmacological inhibition of ERRalpha with XCT790 attenuated insulin-induced glucokinase expression. Taken together, this research identifies glucokinase as a novel target of PGC-1alpha/ERRalpha and underscores the regulatory function of ERRalpha in insulin-dependent enzyme regulation.

Knockdown of CRAD suppresses the growth and promotes the apoptosis of human lung cancer cells via Claudin 4.

Non-small cell lung cancer (NSCLC) is one of the most common causes of cancer-related mortality globally. However, the mechanism underlying NSCLC is not fully understood. Here, we investigated the role of cancer-related regulator of actin dynamics (CRAD) in NSCLC. We showed that CRAD was up-regulated in human NSCLC tissues and lung cancer cell lines. Lentivirus-mediated knockdown of CRAD repressed the proliferation and colony growth of A549 and H1299 cells. Apoptosis was enhanced by CRAD silencing in both cells, implicating that CRAD might maintain the survival of lung cancer cells. Microarray and bioinformatic assay revealed that CRAD directly or indirectly regulated diverse genes, including those involved in cell cycle and DNA damage repair. qRT-PCR and Western blot results confirmed the dysregulated genes as shown in microarray analysis. Claudin 4 was up-regulated in CRAD silenced A549 cells. The knockdown of Claudin 4 blocked the effects of CRAD on the expression of cell cycle and apoptosis effectors and enhanced the viability of A549 cells with CRAD down-regulation. Taken together, our findings demonstrate that CRAD acts as an oncogene in NSCLC at least partly through repressing Claudin 4.

[Research advances in Sirt1 gene].

As the most homologic homologue of silent information regulator 2 of yeast, Sirt1 gene is extensively expressed in mature tissues, and is rich in early embryo and reproductive cells. It is involved in the regulation of gene transcription, energy metabolism and cell aging. It promotes fat mobilization in adipocytes and glucose production in liver and regulates insulin secretion in islet beta cell. Furthermore, Sirt1 gene is an essential endogenous apoptosis inhibitor. In future, it may be used as new drug targets or applied in other disease management modalities.

[Current situation researching of methylation in tumor].

The disorders of DNA and histone methylation have a close relationship with the development and progression of tumors. Epigenetic regulation is critical in maintaining the stability and integrity of the expression profiles of different cell types by modifying DNA methylation and histone methylation. However, the abnormal changes of methylation often result in the development and progression of tumors. This review summarized the theory of tumor genomic and histone methylation, detection methods of methylation and their applications, and the clinical application of methylation as biological markers and drug targets.

Dexamethasone-induced Krüppel-like factor 9 expression promotes hepatic gluconeogenesis and hyperglycemia.

Chronic glucocorticoid therapy has serious side effects, including diabetes and fatty liver. However, the molecular mechanisms responsible for steroid-induced diabetes remain largely enigmatic. Here, we show that hepatic Krüppel-like factor 9 (Klf9) gene expression is induced by dexamethasone and fasting. The overexpression of Klf9 in primary hepatocytes strongly stimulated Pgc1a gene expression through direct binding to its promoter, thereby activating the gluconeogenic program. However, Klf9 mutation abolished the stimulatory effect of dexamethasone on cellular glucose output. Adenovirus-mediated overexpression of KLF9 in the mouse liver markedly increased blood glucose levels and impaired glucose tolerance. Conversely, both global Klf9-mutant mice and liver-specific Klf9-deleted mice displayed fasting hypoglycemia. Moreover, the knockdown of Klf9 in the liver in diabetic mouse models, including ob/ob and db/db mice, markedly lowered fasting blood glucose levels. Notably, hepatic Klf9 deficiency in mice alleviated hyperglycemia induced by chronic dexamethasone treatment. These results suggest a critical role for KLF9 in the regulation of hepatic glucose metabolism and identify hepatic induction of KLF9 as a mechanism underlying glucocorticoid therapy-induced diabetes.

Involvement of KLF11 in hepatic glucose metabolism in mice via suppressing of PEPCK-C expression.

BACKGROUND: Abnormal hepatic gluconeogenesis is related to hyperglycemia in mammals with insulin resistance. Despite the strong evidences linking Krüppel-like factor 11 (KLF11) gene mutations to development of Type 2 diabetes, the precise physiological functions of KLF11 in vivo remain largely unknown. RESULTS: In current investigation, we showed that KLF11 is involved in modulating hepatic glucose metabolism in mice. Overexpression of KLF11 in primary mouse hepatocytes could inhibit the expression of gluconeogenic genes, including phosphoenolpyruvate carboxykinase (cytosolic isoform, PEPCK-C) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), subsequently decreasing the cellular glucose output. Diabetic mice with overexpression of KLF11 gene in livers significantly ameliorated hyperglycemia and glucose intolerance; in contrast, the knockdown of KLF11 expression in db/m and C57BL/6J mice livers impaired glucose tolerance. CONCLUSIONS: Our data strongly indicated the involvement of KLF11 in hepatic glucose homeostasis via modulating the expression of PEPCK-C.

Tff3, as a novel peptide, regulates hepatic glucose metabolism.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder strongly associated with hepatic glucose intolerance and insulin resistance. The trefoil peptides are a family of small regulatory proteins and Tff3 is widely expressed in multiple tissues including liver. But the roles of Tff3 in regulation of glucose metabolism and insulin sensitivity in liver remain unclear. Here we show that the hepatic Tff3 expression levels were decreased in ob/ob and high-fat diet-induced obese mice. Overexpression of Tff3 in primary mouse hepatocytes inhibited the expression of gluconeogenic genes, including G6pc, PEPCK and PGC-1α, subsequently decreasing cellular glucose output. GTT and ITT experiments revealed that adenovirus-mediated overexpression of Tff3 in diabetic or obese mice improved glucose tolerance and insulin sensitivity. Collectively, our results indicated that Tff3 peptides are involved in glucose homeostasis and insulin sensitivity, providing a promising peptide on new therapies against the metabolic disorders associated with T2DM.

PGC-1 beta-regulated mitochondrial biogenesis and function in myotubes is mediated by NRF-1 and ERR alpha.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) coactivator-1 beta (PGC-1 beta) is a well-established regulator of the beta-oxidation of fatty acids and the oxidative phosphorylation in mitochondria. However, the underlying mechanism of PGC-1 beta action remains elusive. This study reveals that PGC-1 beta is highly induced during myogenic differentiation and knockdown of endogenous PGC-1 beta by siRNA leads to a decrease in the expression of several mitochondria-related genes. In consistence, the over-expression of PGC-1 beta stimulates its target genes such as cytochrome c, ATP synthase beta and ALAS-1 by its interaction with two transcriptional factors, NRF-1 and ERR alpha. The deletion or mutation of NRF-1 and/or ERR alpha binding sites in target gene promoters attenuates their activation by PGC-1 beta. Moreover, inhibition of NRF-1 or ERR alpha by siRNA ablated the aforesaid function of PGC-1 beta and compromised the oxidative phosphorylation and mitochondrial biogenesis. Taken together, these results confirm the direct interaction of NRF-1 and ERR alpha with PGC-1 beta, and their participation in mitochondrial biogenesis and respiration.