Regulation of hepatic micro RNA expression by hepatocyte nuclear factor 4 alpha

AIMTo uncover the role of hepatocyte nuclear factor 4 alpha (HNF4α) in regulating hepatic expression of microRNAs. METHODSMicroarray and real-time PCR were used to determine hepatic expression of microRNAs in young-adult mice lacking Hnf4α expression in liver (Hnf4α-LivKO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4α, RNA polymerase-II, and histone modifications to loci of microRNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4α on the promoters of mouse and human microRNAs as well as effects of microRNAs on the untranslated regions (3’UTR) of two genes in human hepatoma cells. RESULTSMicroarray data indicated that most microRNAs remained unaltered by Hnf4α deficiency in Hnf4α-LivKO mice. However, certain liver-predominant microRNAs were down-regulated similarly in young-adult male and female Hnf4α-LivKO mice. The down-regulation of miR-101, miR-192, miR-193a, miR-194, miR-215, miR-802, and miR-122 as well as induction of miR-34 and miR-29 in male Hnf4α-LivKO mice were confirmed by real-time PCR. Analysis of public chromatin immunoprecipitation-sequencing data indicates that HNF4α directly binds to the promoters of miR-101, miR-122, miR-194-2/miR-192 and miR-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4α markedly activated the promoters of mouse and human miR-101b/miR-101-2 and the miR-194/miR-192 cluster. Additionally, miR-192 and miR-194 significantly decreased activities of luciferase reporters for the 3’UTR of histone H3F3 and chromodomain helicase DNA binding protein 1 (CHD1), respectively, suggesting that miR-192 and miR-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1. CONCLUSIONHNF4α is essential for hepatic basal expression of a group of liver-enriched microRNAs, including miR-101, miR-192, miR-193a, miR-194 and miR-802, through which HNF4α may play a major role in the post-transcriptional regulation of gene expression and maintenance of the epigenome in liver.

Expression of hepatocyte nuclear factor 4 alpha,wingless-related integration site,andβ-catenin in clinical gastric cancer

BACKGROUND Gastric cancer(GC)is the second most common cause of cancer-related deaths worldwide.Hepatocyte nuclear factor 4 alpha(HNF4α)that belongs to the nuclear hormone receptor superfamily,is overexpressed in GC tissues,and might be involved in the development of GC by regulating its downstream winglessrelated integration site(WNT)/β-catenin signaling.AIM To clarify the expression of HNF4α/WNT5a/β-catenin signaling proteins in clinical GC tissues.METHODS We immunohistochemically stained pathological blocks of GC and matched paracancerous tissues.The intensity of HNF4α,WNT5a andβ-catenin staining in the tumor cells was determined according to cell rates and staining intensity.The correlations between GC and HNF4α,WNT5a,andβ-catenin expression using chisquare and paired chi-square tests.Relationships between double-positive HNF4αand WNT5a expression and types of gastric tumor tissues were assessed using regression analysis.Correlations between HNF4αand WNT5a expression at the RNA level in GC tissues found in the TCGA database were analyzed using Pearson correlation coefficients.RESULTS We found more abundant HNF4αand WNT5a proteins in GC,especially in mucinous adenocarcinoma and mixed GC than in adjacent tissues(P<0.001).Low and high levels of cytoplasmicβ-catenin respectively expressed in GC and adjacent tissues(P<0.001)were not significantly associated with pathological parameters.CONCLUSION The expressions of HNF4αand WNT5a could serve as early diagnostic biomarkers for GC.

Facilitating effects of berberine on rat pancreatic islets through modulating hepatic nuclear factor 4 alpha expression and glucokinase activity

AIM： To observe the effect of berberine on insulin secretion in rat pancreatic islets and to explore its possible molecular mechanism. METHODS： Pdmary rat islets were isolated from male Sprague-Dawley rats by collagenase digestion and treated with different concentrations （1, 3, 10 and 30 μmol/L） of berberine or 1 μmol/L Glibenclamide （GB） for 24 h. Glucose-stimulated insulin secretion （GSIS） assay was conducted and insulin was determined by radioimmunoassay. 3-（4,5-Dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （MTT） assay was performed to evaluate cytotoxicity. The mRNA level of hepatic nuclear factor 4 alpha （HAIF4α） was determined by reverse transcription polymerase chain reaction （RT-PCR）. Indirect immunofluorescence staining and Western blot analysis were employed to detect protein expression of HNF4α in the islets. Glucokinase （GK） activity was measured by spectrophotometric method. RESULTS： Berberine enhanced GSIS rather than basal insulin secretion dose-dependently in rat islets and showed no significant cytotoxicity on islet cells at the concentration of 10 μmol/L. Both mRNA and protein expressions of HNF4α were up-regulated by berberine in a dose-dependent manner, and GK activity was also increased accordingly. However, GB demonstrated no regulatory effects on HNF4α expression or GK activity. CONCLUSION： Berberine can enhance GSIS in rat islets, and probably exerts the insulinotropic effect via a pathway involving HNF4α and GK, which is distinct from sulphonylureas （SUs）.

转录因子HNF1A、HNF4A和FOXA2调节肝细胞蛋白质N-糖基化

Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

The interplay between hepatocyte nuclear factor 4α(HNF4α)and cholesterol sulfotransferase(SULT2B1b)in hepatic energy homeostasis

The nuclear receptor hepatocyte nuclear factor 4alpha(HNF4α)plays a critical role in the regulation of metabolic homeostasis,including glucose homeostasis.Sulfotransferases(SULTs)catalyze the transfer of a sulfate group from 3-phosphoadenosine 5-phosphosulfate(PAPS)to an acceptor molecule.Sulfonation plays an essential role in regulating the chemical and functional homeostasis of endogenous and exogenous molecules.Among SULTs,the cholesterol sulfotransferase 2B1b(SULT2B1b)preferentially catalyzes the sulfoconjugation of cholesterol and oxysterols to form cholesterol sulfate and oxysterol sulfates.Hepatic gluconeogenesis represents a critical component of energy metabolism.Although there have been reviews on the regulation of glucose homeostasis by HNF4a,the interplay between HNF4a and SULT2B1b in hepatic glucose homeostasis remains scattered.In this review,we intend to provide an overview on how HNF4a functionally cross-talks with SULT2B1b to regulate hepatic glucose homeostasis and whether the HNF4a-SULT2B1b axis represents a novel therapeutic target for the management of metabolic liver disease and metabolic syndrome.