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Background: We estimated the diagnostic and prognostic value of serum angiopoietin-2 (Ang-2) in liver cancer patients., Methods: Tissue Ang-2 was measured using immunohistochemistry (IHC). Cell localization of Ang-2 was tested using immunofluorescence (IF). Cell viability and apoptosis were evaluated using MTT and caspase3/7 assays, respectively. Colony-formation was measured using a soft agar assay. Serum Ang-2 was examined using enzyme-linked immunosorbent assay (ELISA) and Western blotting., Results: Ang-2 was up-regulated in liver cancer compared to the levels in normal tissues. Serum Ang-2 concentrations were much higher in liver cancer patients than in healthy individuals and those with chronic liver disease (CLD). Inhibitions of Ang-2 using specific shRNA decreased cell proliferation. Serum Ang-2 decreased significantly after surgery. Serum Ang-2 was positively correlated with serum alpha-fetoprotein (AFP; R=0.375, P=0.005). Receiver operating characteristic (ROC) curves suggested that serum Ang-2 could be used with relatively high sensitivity and specificity in differentiating liver cancer patients from CLD patients or healthy controls, with corresponding AUC values of 0.742 and 0.924, respectively. Serum Ang-2 was negatively correlated with overall survival. Subgroup analysis also showed that Ang-2 retained its prognostic value in overall survival prediction in different risk subgroups., Conclusion: Serum Ang-2 may be a useful tumor marker in predicting liver cancer prognosis., (Copyright © 2017. Published by Elsevier B.V.)

AMOT has been identified as a YAP interactor. However, how AMOT regulates YAP remains unclear and controversy. Here, we identified that besides YAP, AMOT was another Hippo signaling core factor which could be O-GlcNAcylated. Moreover, high glucose (HG) was able to enhance the expression and O-GlcNAcylation of AMOT. We also found that HG stimulated nuclear accumulation, transcription activity, interaction with transcription factor and transcription of target genes of YAP via AMOT, while AMOT acted as a suppressor of YAP in normal glucose level. Finally, we observed the upregulation and nuclear accumulation of AMOT and YAP in Streptozocin (STZ) induced high glucose mice. Collectively, we have uncovered that AMOT acts as a YAP stimulator in high glucose level. Targeting the aberrantly regulated core factors in Hippo pathway might be a more effective therapeutic approach for liver cancer associated with possibly diabetes., (Copyright © 2017. Published by Elsevier Inc.)

The Wnt pathway is crucial for animal development, as well as tumor formation. Understanding the regulation of Wnt signaling will help to elucidate the mechanism of the cell cycle, cell differentiation and tumorigenesis. It is generally accepted that in response to Wnt signals, β-catenin accumulates in the cytoplasm and is imported into the nucleus where it recruits LEF/TCF transcription factors to activate the expression of target genes. In this study, we report that human NRAGE, a neurotrophin receptor p75 (p75NTR) binding protein, markedly suppresses the expression of genes activated by the Wnt pathway. Consistent with this finding, loss of function of NRAGE by RNA interference (RNAi) activates the Wnt pathway. Moreover, NRAGE suppresses the induction of axis duplication by microinjected β-catenin in Xenopus embryos. To our surprise, NRAGE induces nuclear localization of β-catenin and increases its DNA binding ability. Further studies reveal that NRAGE leads to the modification of β-catenin/Arm with O-linked beta-N-acetylglucosamine (O-GlcNAc), and failure of the association between β-catenin/Arm and pygopus(pygo) protein, which is required for transcriptional activation of Wnt target genes. Therefore, our findings suggest a novel mechanism for regulating Wnt signaling., (Copyright © 2017 Elsevier Inc. All rights reserved.)

MicroRNAs are small non-coding RNAs with 18-22 nucleotides in length and have been proposed to function in various biological processes by targeting genes for post-transcriptional degradation via their 3' untranslated region. Moreover, they have been suggested to improve the chemosensitivity in a panel of tumors. However, the biological functions of microRNA-127-3p in esophageal carcinogenesis are still enigmatic. Thus, in the study, we firstly analyzed the roles of microRNA-127-3p in regulating the growth of esophageal cancer cells both in vitro and in vivo. Afterwards, using the microRNA-targeted gene prediction software and the dual-luciferase reporter assays, we confirmed that microRNA-127-3p specifically reduced the expression of X-ray repair complementing defective repair in Chinese hamster cells 3, one of RAD51 recombinase paralogs, at both mRNA and protein levels. Furthermore, using the homologous recombination repair and non-homologous end joining repair reporter systems, we found that microRNA-127-3p specifically compromised the homologous recombination repair and significantly increased DNA double strand breaks in cells. Besides, it statistically increased the chemosensitivity of esophageal cancer cells to a novel phenanthroline-dione derivative in vivo by mechanistically impairing the recruitment of RAD51 to the damage sites. In summary, our findings not only suggest that microRNA-127-3p can be used as a predictor for evaluating the development of esophageal carcinoma, but also show that it can be used to increase the chemosensitivity of esophageal cancer patients to the phenanthroline-dione derivative, which might be a potential anticancer candidate in the future., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: We evaluated the diagnostic value of serum-CD166 in patients with hepatocellular carcinoma (HCC)., Methods: Tissue-CD166 was measured using immunohistochemistry. Cell proliferation and migration were evaluated using MTT and Transwell assays, respectively. Serum-CD166 was examined using ELISA and western blotting., Results: CD166 was up-regulated in HCC compared to those in normal liver tissues. Cell proliferation was positively correlated and cell migration was negatively correlated with endogenous CD166 expression in HCC cells. CD166 inhibition using specific shRNA decreased cell proliferation but increased cell migration. Serum CD166 concentrations were much higher in HCC than in colon cancer, hepatitis B, hepatitis C, cirrhosis, gastric cancer, breast cancer, lung cancer and healthy individuals. Serum CD166 also decreased dramatically after curative surgery. A positive correlation was found between serum CD166 and AFP (R=0.7141, p=0.000). Serum CD166 was also positively correlated with γ-GT, bile acid, ALT, AST, and ALP but was negatively correlated with Alb and pre-Alb. The area under the receiver operating characteristic curve for serum-CD166 was 0.9860, which was better than AFP (AUC-ROC, 0.9354) for the differentiation of HCC patients from healthy individuals, with a cut-off of 261 ng/ml (sensitivity: 100.00%, specificity: 89.41%)., Conclusion: Serum CD166 is a novel diagnostic tumor marker for HCC., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cluster of differentiation 166 (CD166) is critical for liver cancer cell survival. Our previously study demonstrated that CD166 exerts its anti-apoptotic role through interaction with YAP in liver cancer. However, the interaction between CD166 and other cell surface molecules remains unclear in liver cancer cells. In the current study, we found that both mRNA and protein of CD44 expression was significantly inhibited by knocking-down CD166. Moreover, CD166 affected-CD44 expression is dependent of transcription via blocking NF-κB pathway. On the contrary, CD44 promoted up-regulation of CD166 mRNA and protein. And it may be through E3 ubiquitin ligases COP1 and UBC3 to regulate CD166 protein degradation. Collectively, these results suggest that CD166 and CD44 play important roles in liver cancer development. Therefore, CD166 may develop as a potential therapeutic molecule target for the treatment of liver cancer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Yes-associated protein (YAP), the downstream effector of Hippo signaling pathway as well as c-Myc has been linked to hepatocarcinogenesis. However, little is known about whether and how YAP and c-Myc interacts with each other. In this study, we find YAP-c-Myc interaction is critical for liver cancer cell both in vitro and in vivo. Moreover, both c-Myc and YAP proteins are closely correlated in human liver cancer samples. Mechanistically, YAP promotes c-Myc transcriptional output through c-Abl. By contrast, c-Myc enhances protein expression independent of transcription. Taken together, our study uncovers a novel positive auto-regulatory feedback loop underlying the interaction between YAP and c-Myc in liver cancer, suggesting YAP and c-Myc links Hippo/YAP and c-Myc pathways, and thus may be helpful in the development of effective diagnosis and treatment strategies against liver cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Bone formation is a developmental process requiring the differentiation of mesenchymal stem cells into osteoblasts. It is established that Runx2 tightly regulates osteoblast differentiation and bone formation. Fos-related antigen Fra-1 is an essential factor for bone formation. Current evidence does not support a relationship between Fra-1 and Runx2 in osteogenesis. Here, we explored the possibility that Runx2 regulates Fra-1 expression during osteogenic differentiation of C2C12 myogenic progenitor cells. Expression of Fra-1 was induced rapidly after activation of Runx2 in a Tet-on stable C2C12 cell-line (C2C12/Runx2(Dox) sub-line). Transient transfection assay showed that Runx2 activates Fra-1 promoter-reporter activity, suggesting that Fra-1 may be a direct target of Runx2. To determine the minimal region of the Fra-1 promoter that was activated by Runx2, a series of Fra-1 promoter deletion constructs were made. By transient transfection assay, we defined the minimal region to the proximal 342 bp (-84 to +258). Two potential Runx2-binding sites (at positions +139 and +208) were predicted within this region. Mutation of the Runx2 motif at position +208 significantly decreased Fra-1 promoter activity compared to wild type, whereas mutation of Runx2 at position +139 had no effect. Electrophoretic mobility shift assay (EMSA) demonstrated the existence of one atypical Runx2-binding element at position +208, and chromatin immunoprecipitation (ChIP) assay revealed that Runx2 bound to the native Fra-1 promoter in vivo via this site. Finally, forced expression of Fra-1 resulted in upregulation of alkaline phosphatase (ALP), a marker of early osteoblast differentiation. Together, these results indicate that Fra-1 is a direct target of Runx2 during osteogenic differentiation of C2C12 myogenic progenitor cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

ADAM23 modulates many cellular functions, alteration of expression causes a number of tumor types; however, the mechanisms controlling ADAM23 expression remain unknown. Here we have identified a SP1 binding site (-202/-190) that binds SP1 at the proximal promoter of human ADAM23 gene; furthermore, serum deprivation enhances open chromatin accessibility and help expose the SP1 binding site; finally, SP1 binding recruits RNA polymerase II, which in turn results in upregulation of endogenous ADAM23 expression. Therefore, the present study delineates the fundamental elements of a core promoter structure that will be helpful for future studies of the regulation of ADAM23 gene., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Aims: The identification of transcription factors that regulate the transcription of the fibroblast growth factor-2 (FGF-2) gene has facilitated the understanding of the etiology of cardiovascular diseases. The purpose of this study was to determine the molecular mechanism underlying the activation of FGF-2 gene transcription in cardiomyocytes from neonatal rats., Main Methods: To identify the factors involved in cardiac expression of FGF-2, we used transient transfections in neonatal rat cardiomyocytes coupled with electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) analyses., Key Findings: Deletion analyses showed that the region between -16 and +59 was essential for maximal FGF-2 promoter activity. Three putative stimulating protein 1 (Sp1) regulatory sites located at positions -3, +14, and +27 were predicted within this region by computer analysis. EMSA showed the existence of two atypical G-rich Sp1-binding elements located at positions -3 and +14. Mutation of these two sites resulted in a significant decline in FGF-2 promoter activity compared to wild type promoter activity. Combinatorial mutation of these sites reduced the promoter activity to background levels. Mutation of the Sp1 motif at +27 did not affect promoter activity. Lastly, ChIP analyses revealed that Sp1 binds to the FGF-2 promoter region in vivo., Significance: These results indicate that expression of FGF-2 in neonatal rat cardiomyocytes is associated with Sp1 binding to the FGF-2 promoter.

To date, numerous microRNAs (miRNAs) have been discovered. However, the function of these miRNAs is largely unknown. While our knowledge of miRNA post-transcriptional processing has greatly expanded in recent years, we have a limited understanding of the regulation and transcription of miRNA genes. In this study, we characterized two BMP-2 upregulated miRNAs, miR-24-1 and miR-31, in mesenchymal stem cells and showed their opposing function in controlling cellular proliferation, and adipogenesis. Furthermore, we are the first to identify and characterize mouse intronic miR-23b~27b~24-1 and intergenic miR-31 genes. Moreover, we found that pri-miR-23b, pri-miR-27b, and pri-miR-24-1 are transcribed independently and their expression profiles are unique when cells are treated with BMP-2, even though they are located closely together.

In this study, we identify and characterize the promoter of the mouse Gas6 gene, a negative regulator of chondrogenic differentiation. We identified two highly conserved regions within the core Gas6 promoter, which are conserved among mouse, human, and rat Gas6 genes, named A-BOX and B-BOX. Basal transcriptional activity was significantly reduced after deletion of either the A-BOX or the latter half of the B-BOX. After treatment with BMP-2 for 3 days, putative A-BOX Binding Factor(s) B (ABFB) and B-BOX Binding Factor(s) (BBF) were unable to bind to their specific motifs in C3H10T1/2 cells, compared with untreated control cells. In addition, we confirmed binding of an NF-Y site within the core promoter region of mouse Gas6 gene. Taken together, these observations provide insight into the mechanism by which Gas6 is downregulated during mesenchymal stem cells differentiation into chondrocytes.