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Xu J.,Henan University of Technology | Wong C.-W.,NeuMed Pharmaceuticals Ltd
Methods in Molecular Biology | Year: 2013

MicroRNAs (miRNAs) are a class of ~22-nucleotide endogenous noncoding RNAs which regulate target gene expression via repressing translation or promoting mRNAs degradation. Any individual mammalian miRNA often has more than a hundred predicted mRNA targets and that close to one thirds of all mRNA transcripts bear one or more conserved miRNA binding sites in their 3 ¢ -untranslated region. Enrichment analysis of miRNA targets has become a standard technique to elucidating hierarchical functions of miRNAs in gene regulatory networks. In this protocol, we discuss analytical methods and use of computational tools in a step-by-step manner. Important details are also provided to help researchers choose more appropriate tools for a given type of analysis. Available Web-based resources for enrichment analysis of miRNA targets are summarized. © Springer Science+Business Media, LLC 2013.

Gao M.,CAS Guangzhou Institute of Biomedicine and Health | Wang J.,CAS Guangzhou Institute of Biomedicine and Health | Lu N.,CAS Guangzhou Institute of Biomedicine and Health | Fang F.,CAS Guangzhou Institute of Biomedicine and Health | And 2 more authors.
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2011

Growth factor activates mitogen-activated protein kinase kinases to promote cell growth. Mitochondrial biogenesis is an integral part of cell growth. How growth factor regulates mitochondrial biogenesis is not fully understood. In this study, we found that mitochondrial mass was specifically reduced upon serum starvation and induced upon re-feeding with serum. Using mitogen-activated protein kinase kinases inhibitor U0126, we found that the mRNA expression levels of ATP synthase, cytochrome-C, mitochondrial transcription factor A, and mitofusin 2 were reduced. Since the transcriptional levels of these genes are under the control of peroxisome proliferator-activated receptor γ coactivator-1α and -1β (PGC-1α and PGC-1β), we examined and found that only the mRNA and protein levels of PGC-1β were suppressed. Importantly, over-expression of PGC-1β partially reversed the reduction of mitochondrial mass upon U0126 treatment. Thus, we conclude that mitogen-activated protein kinase kinases direct mitochondrial biogenesis through selectively inducing PGC-1β expression. © 2011 Elsevier B.V.

Lu N.,CAS Guangzhou Institute of Biomedicine and Health | Wang W.,CAS Guangzhou Institute of Biomedicine and Health | Wang W.,Oregon Health And Science University | Liu J.,CAS Guangzhou Institute of Biomedicine and Health | Wong C.-W.,NeuMed Pharmaceuticals Ltd
Cellular Signalling | Year: 2011

Members of the protein kinase C (PKC) family have been implicated in controlling cell proliferation, differentiation, and motility. Many of these processes are energy demanding. How PKCs affect mitochondrial function to regulate energy production is not well defined. Using an inhibitor Gö6983 with broad specificity, we found that inhibiting PKCs reduced mitochondrial mass and altered mitochondrial function characterized by elevations in mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels. These alterations indicated that Gö6983 suppressed the activities of mitochondrial regulators such as estrogen-related receptor α (ERRα). Indeed, Gö6983 dose-dependently suppressed the expression levels of ERRα-target genes peroxisome proliferator-activated receptor α (PPARα) and medium-chain acyl-CoA dehydrogenase (MCAD). Conversely, PKC activator phorbol ester (PMA) enhanced the expression level of another ERRα-target gene pyruvate dehydrogenase kinase 4 (PDK4). This PMA-mediated induction of PDK4 was blunted by an ERRα inverse agonist XCT-790, suggesting that ERRα plays a role in mediating the effects of PKCs on mitochondrial function. By over-expressing constitutively active forms of PKCs, we found that PKCε preferentially stimulated the transcription activity of ERRα. Through mutating residues on ERRα, we established that this PKCε-induced ERRα activity involves threonine 106, serine 110, and threonine 124 of ERRα. Collectively, these pieces of evidence suggest that ERRα plays an important role down-stream of PKCε to regulate mitochondrial homeostasis. © 2011 Elsevier Inc.

Liu Z.,Nanjing University of Technology | Law W.-K.,NeuMed Pharmaceuticals Ltd | Wang D.,Nanjing University of Technology | Nie X.,Nanjing University of Technology | And 7 more authors.
RSC Advances | Year: 2014

Based upon the discovery of the natural compound andrographolide (1) as a non-steroidal farnesoid X receptor (FXR) antagonist, a series of andrographolide derivatives were designed and synthesized accordingly. Our primary SAR studies demonstrated that 14-phenoxy andrographolide scaffold is an excellent structural pharmacophore for FXR antagonists. Remarkably, 14β-compounds of 12b, 12f and 10g were found to be the most potent FXR antagonists in this work. Structural docking discovered that the phenoxy substitution at the 14-position and the modification at 3,19-positions altered the putative binding positions of small FXR ligands, resulting in their FXR antagonistic activity discrepancy. This journal is © the Partner Organisations 2014.

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