TGR5: A Novel Target for Weight Maintenance and Glucose Metabolism

TGR5, an emerging G protein-coupled receptor, was identified as a membrane receptor for bile acids. The expression of TGR5 and its function are distinct from the previously identified nuclear bile acid receptor, farnesoid X receptor (FXR). These two bile acid receptors complement with each other for maintaining bile acid homeostasis and mediating bile acid signaling. Both receptors are also shown to play roles in regulating inflammation and glucose metabolism. An interesting finding for TGR5 is its role in energy metabolism. The discovery of TGR5 expression in brown adipocyte tissues (BATs) and the recent demonstration of BAT in adult human body suggest a potential approach to combat obesity by targeting TGR5 to increase thermogenesis. We summarize here the latest finding of TGR5 research, especially its role in energy metabolism and glucose homeostasis

MiR-137 Targets Estrogen-Related Receptor Alpha and Impairs the Proliferative and Migratory Capacity of Breast Cancer Cells

ERRα is an orphan nuclear receptor emerging as a novel biomarker of breast cancer. Over-expression of ERRα in breast tumor is considered as a prognostic factor of poor clinical outcome. The mechanisms underlying the dysexpression of this nuclear receptor, however, are poorly understood. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play important roles in tumor initiation and progression. In the present study, we have identified that the expression of ERRα is regulated by miR-137, a potential tumor suppressor microRNA. The bioinformatics search revealed two putative and highly conserved target-sites for miR-137 located within the ERRα 3′UTR at nt 480–486 and nt 596–602 respectively. Luciferase-reporter assay demonstrated that the two predicted target sites were authentically functional. They mediated the repression of reporter gene expression induced by miR-137 in an additive manner. Moreover, ectopic expression of miR-137 down-regulated ERRα expression at both protein level and mRNA level, and the miR-137 induced ERRα-knockdown contributed to the impaired proliferative and migratory capacity of breast cancer cells. Furthermore, transfection with miR-137mimics suppressed at least two downstream target genes of ERRα–CCNE1 and WNT11, which are important effectors of ERRα implicated in tumor proliferation and migration. Taken together, our results establish a role of miR-137 in negatively regulating ERRα expression and breast cancer cell proliferation and migration. They suggest that manipulating the expression level of ERRα by microRNAs has the potential to influence breast cancer progression

HBx inhibits CYP2E1 gene expression via downregulating HNF4α in human hepatoma cells.

CYP2E1, one of the cytochrome P450 mixed-function oxidases located predominantly in liver, plays a key role in metabolism of xenobiotics including ethanol and procarcinogens. Recently, down-expression of CYP2E1 was found in hepatocellular carcinoma (HCC) with the majority to be chronic hepatitis B virus (HBV) carriers. In this study, we tested a hypothesis that HBx may inhibit CYP2E1 gene expression via hepatocyte nuclear factor 4α (HNF4α). By enforced HBx gene expression in cultured HepG2 cells, we determined the effect of HBx on CYP2E1 mRNA and protein expression. With a bioinformatics analysis, we found a consensus HNF-4α binding sequence located on -318 to -294 bp upstream of human CYP2E1 promoter. Using reporter gene assay and site-directed mutagenesis, we have shown that mutation of this site dramatically decreased CYP2E1 promoter activity. By silencing endogenous HNF-4α, we have further validated knockdown of HNF-4α significantly decreased CYP2E1 expression. Ectopic overexpression of HBx in HepG2 cells inhibits HNF-4α expression, and HNF-4α levels were inversely correlated with viral proteins both in HBV-infected HepG2215 cells and as well as HBV positive HCC liver tissues. Moreover, the HBx-induced CYP2E1 reduction could be rescued by ectopic supplement of HNF4α protein expression. Furthermore, human hepatoma cells C34, which do not express CYP2E1, shows enhanced cell growth rate compared to E47, which constitutively expresses CYP2E1. In addition, the significantly altered liver proteins in CYP2E1 knockout mice were detected with proteomics analysis. Together, HBx inhibits human CYP2E1 gene expression via downregulating HNF4α which contributes to promotion of human hepatoma cell growth. The elucidation of a HBx-HNF4α-CYP2E1 pathway provides novel insight into the molecular mechanism underlining chronic HBV infection associated hepatocarcinogenesis

MiR-137 influences the migratory capacity of MDA-MB-231 cells partly through ERRα-WNT11 signaling pathway.

<p>A. re-expression of ERRα (without 3′-UTR) in MDA-MB-231 cells restored the impaired migratory capacity induced by miR-137. MDA-MB-231 cells were co-transfected with indicated RNA oligonucleotides (50 nM) and plasmids (1 µg), and serum starved for 12 hr, followed by assessment of cell invasion and viability. Error bars: SD; *: p<0.05; **: P<0.01; ***: P<0.0001. B. re-expression of ERRα (without 3′-UTR) in MDA-MB-231 cells reversed the decrease of WNT11 expression induced by miR-137. MDA-MB-231 cells were co-transfected with indicated RNA oligonucleotides (50 nM) and plasmids (1 µg). 48 hr after transfection, protein and mRNA levels of WNT11 and ERRα were assayed using western bolt and qRT-PCR respectively. WNT11 or ERRα mRNA expression was normalized to β-actin mRNA expression. The relative level of WNT11 or ERRα determined using the 2-^△△CT method. Data are representative of three independent experiments performed in duplicate. Error bars: SD; **: P<0.01; ***: P<0.0001.</p

Identification of two highly conserved miR-137 target sites within the ESRRA 3′UTR.

<p>A. Schematic representation of the ERRα (ESRRA) mRNA with two putative sites (A and B) targeted by miR-137. B. Sequence alignment of predicted miR-137 target sites located within ESRRA 3′UTR showing high conservation among different species. The sequence of miR-137 target sites in ESRRA 3′UTR is shown in underlined. C. Luciferase reporter assay to verify activity of miR-137 upon the consensus miR-137 target site. HepG2 cells were transfected with Empty reporter plasmids, luciferase constructs containing perfect match miR-137 target site (miR-137 target) or mismatch miR-137 target site (△miR-137 target) and either miR-137 mimcs or NC oligos. Luciferase activity was determined 24 hr after transfection. Relative luciferase expression (firefly normalized to Renilla) values are the ratio of miR-137-treated reporter vector compared with the same NC oligos-treated reporter vector. Data are representative of at least three independent experiments. Error bars: SD. ***: P<0.0001. D. Luciferase reporter assay to evaluate the interaction between miR-137 and 3′-UTR of ESRRA. HepG2 cells were transfected with luciferase constructs containing wild-type (WT 3′UTR) or deletion mutated ESRRA 3′UTR (mutant A, mutant B and mutant C) and either miR-137 mimcs or NC oligos. Luciferase activity was determined 24 hr after transfection. Relative luciferase expression (firefly normalized to Renilla) values are the ratio of miR-137-treated reporter vector compared with the same NC oligos-treated reporter vector. Data are representative of three independent experiments. Error bars: SD. *: p<0.05, ***: P<0.0001.</p

Ectopic transfection of miR-137 regulates the endogenous ERRα expression level.

<p>A. Western blot analysis for ERRα protein level and qRT-PCR analysis for ERRα mRNA level in SK-BR-3 cells 48 hr after transfection regent treatment (mock) or transfection with indicated RNA oligonucleotides (50 nM). B. Western blot analysis for ERRα protein level and qRT-PCR analysis for ERRα mRNA level in SK-BR-3 cells 48 hr after transfection regent treatment (mock) or cotransfection with equal amount of indicated RNA oligonucleotides. ERRα mRNA expression was normalized to β-actin mRNA expression. The relative level of ERRα expression determined using the 2-^△△CT method. Data are representative of three independent experiments performed in triplicate. Error bars: SD; *: p<0.05; ***: P<0.0001.</p

Primers used for PCR.

<p>The recognition sites of restriction endonuclease are underlined.</p

The expression of ERRα downstream target gene CCNE1 is regulated by miR-137.

<p>A. Western blot analysis for ERRα and CylinE1 protein level and qRT-PCR analysis for CCNE1 mRNA level in SK-BR-3 cells 48 hr after DMSO or XCT-790 treatment. B. Western blot analysis for CylinE1 protein level and qRT-PCR analysis for CCNE1 mRNA level in SK-BR-3 cells 48 hr after transfection regent treatment (mock) or transfection with NC oligos or si-ERRα. C. Western blot analysis for CylinE1 protein level and qRT-PCR analysis for CCNE1 mRNA level in SK-BR-3 48 hr after transfection regent treatment (mock) or co-transfection with equal amount of indicated RNA oligonucleotides. CCNE1 mRNA expression was normalized to β-actin mRNA expression. The relative level of CCNE1 mRNA was determined using the 2-^△△CT method. Data are representative of three independent experiments performed in triplicate. Error bars: SD; *: p<0.05; **: P<0.01; ***: P<0.0001.</p