Promoter and enhancer RNAs regulate chromatin reorganization and activation of miR-10b/HOXD locus, and neoplastic transformation in glioma.

miR-10b is silenced in normal neuroglial cells of the brain but commonly activated in glioma, where it assumes an essential tumor-promoting role. We demonstrate that the entire miR-10b-hosting HOXD locus is activated in glioma via the cis-acting mechanism involving 3D chromatin reorganization and CTCF-cohesin-mediated looping. This mechanism requires two interacting lncRNAs, HOXD-AS2 and LINC01116, one associated with HOXD3/HOXD4/miR-10b promoter and another with the remote enhancer. Knockdown of either lncRNA in glioma cells alters CTCF and cohesin binding, abolishes chromatin looping, inhibits the expression of all genes within HOXD locus, and leads to glioma cell death. Conversely, in cortical astrocytes, enhancer activation is sufficient for HOXD/miR-10b locus reorganization, gene derepression, and neoplastic cell transformation. LINC01116 RNA is essential for this process. Our results demonstrate the interplay of two lncRNAs in the chromatin folding and concordant regulation of miR-10b and multiple HOXD genes normally silenced in astrocytes and triggering the neoplastic glial transformation.

Thermogenesis-independent metabolic benefits conferred by isocaloric intermittent fasting in ob/ob mice.

Intermittent fasting (IF) is an effective dietary intervention to counteract obesity-associated metabolic abnormalities. Previously, we and others have highlighted white adipose tissue (WAT) browning as the main underlying mechanism of IF-mediated metabolic benefits. However, whether IF retains its efficacy in different models, such as genetically obese/diabetic animals, is unknown. Here, leptin-deficient ob/ob mice were subjected to 16 weeks of isocaloric IF, and comprehensive metabolic phenotyping was conducted to assess the metabolic effects of IF. Unlike our previous study, isocaloric IF-subjected ob/ob animals failed to exhibit reduced body weight gain, lower fat mass, or decreased liver lipid accumulation. Moreover, isocaloric IF did not result in increased thermogenesis nor induce WAT browning in ob/ob mice. These findings indicate that isocaloric IF may not be an effective approach for regulating body weight in ob/ob animals, posing the possible limitations of IF to treat obesity. However, despite the lack of improvement in insulin sensitivity, isocaloric IF-subjected ob/ob animals displayed improved glucose tolerance as well as higher postprandial insulin level, with elevated incretin expression, suggesting that isocaloric IF is effective in improving nutrient-stimulated insulin secretion. Together, this study uncovers the insulinotropic effect of isocaloric IF, independent of adipose thermogenesis, which is potentially complementary for the treatment of type 2 diabetes.

Adipose Tissue and Modulation of Hypertension.

PURPOSE OF REVIEW: Obesity is a major risk factor for the development of hypertension (HTN), a leading cause of cardiovascular morbidity and mortality. Growing body of research suggests that adipose tissue function is directly associated with the pathogenesis of obesity-related HTN. In this review, we will discuss recent research on the role of adipose tissue in blood pressure (BP) regulation and activation of brown adipose tissue (BAT) as a potentially new therapeutic means for obesity-related HTN. RECENT FINDINGS: Adipose tissue provides mechanical protection of the blood vessels and plays a role in regulation of vascular tone. Exercise and fasting activate BAT and induce browning of white adipose tissue (WAT). BAT-secreted FGF21 lowers BP and protects against HTN. Browning of perivascular WAT improves HTN. New insights on WAT browning and BAT activation can open new avenues of potential therapeutic interventions to treat obesity-related HTN.

Regulation of Cell Cycle Associated Genes by microRNA and Transcription Factor.

Cell cycle is a complex process and regulated at transcriptional, post-transcriptional and posttranslational levels. Large numbers of genes are implicated in the process. Abnormality at any stage of cell cycle may lead to diseases including cancer. To gain global view of genes associated with cell cycle, their regulation by transcription factors and microRNAs, we collected genes related to cell cycle from different databases. Experimentally validated targets of microRNAs are collected from miRTarbase. Transcription factors that bind to upstream sequences of cell cycle associated genes and microRNA genes were collected from published papers. We collected 3028 genes associated with cell cycle. These proteins belong to different protein classes like nucleic acid binding (594 proteins), transcription factors (305 proteins), cytoskeletal (232 proteins), kinases (174 proteins), phosphatase (111 proteins) and chaperones (84 proteins). Among 3028 cell cycle associated genes, 2125 genes are validated targets of 424 microRNAs; CDKN1A is a target of 46 miRNAs and miR-335 targets 301 genes. About 100 transcription factors had binding sites at potential promoter regions of 2722 genes and 329 microRNAs that target cell cycle associated genes. We presented the largest numbers of cell cycle associated genes. Many transcription factors regulate both cell cycle associated genes and the miRNAs that target cell cycle associated genes. These resources will be utilized to identify the co-regulation of cell cycle associated genes by transcription factors and miRNAs and to test specific hypothesis for cell cycle regulation and its alteration in different diseases.

Delayed Cell Cycle Progression in STHdh(Q111)/Hdh(Q111) Cells, a Cell Model for Huntington's Disease Mediated by microRNA-19a, microRNA-146a and microRNA-432.

Several indirect evidences are available to indicate that abnormalities in cell cycle may contribute to pathogenesis of Huntington's disease (HD). Here, we show that the cell cycle progression in STsdh(Q111)/Hdh(Q111)cells, a cell model of HD, is delayed in S and G2-M phases compared to control STHdhQ7/HdhQ7cells. Expression of 17 genes, like PCNA and CHEK1, was increased in STHdh(Q111)/Hdh(Q111)cells. Increased expressions of PCNA, CHEK1 and CCNA2, and an enhanced phosphorylation of Rb1 were observed in primary cortical neurons expressing mutant N-terminal huntingtin (HTT), R6/2 mice and STHdh(Q111)/Hdh(Q111) cells. This increase in the expressions of PCNA, CHEK1 and CCNA2 was found to be the result of decreased expressions of miR-432, miR-146a, and (miR-19a and miR-146a), respectively. Enhanced apoptosis was observed at late S phase and G2-M phase in STHdh(Q111)/Hdh(Q111)cells. Exogenous expressions of these miRNAs in STHdh(Q111)/Hdh(Q111) cells rescued the abnormalities in cell cycle and apoptosis. We also observed that inhibitors of cell cycle could decrease cell death in a cell model of HD. Based on these results obtained in cell and animal model of HD, we propose that inhibition of cell cycle either by miRNA expressions or by using inhibitors could be a potential approach for the treatment of HD.

MicroRNA-432 contributes to dopamine cocktail and retinoic acid induced differentiation of human neuroblastoma cells by targeting NESTIN and RCOR1 genes.

MicroRNA (miRNA) regulates expression of protein coding genes and has been implicated in diverse cellular processes including neuronal differentiation, cell growth and death. To identify the role of miRNA in neuronal differentiation, SH-SY5Y and IMR-32 cells were treated with dopamine cocktail and retinoic acid to induce differentiation. Detection of miRNAs in differentiated cells revealed that expression of many miRNAs was altered significantly. Among the altered miRNAs, human brain expressed miR-432 induced neurite projections, arrested cells in G0-G1, reduced cell proliferation and could significantly repress NESTIN/NES, RCOR1/COREST and MECP2. Our results reveal that miR-432 regulate neuronal differentiation of human neuroblastoma cells.

MicroRNA-124 targets CCNA2 and regulates cell cycle in STHdh(Q111)/Hdh(Q111) cells.

Mutation in huntingtin (HTT) gene causes Huntington's disease (HD). Expression of many micro RNAs is known to alter in cell, animal models and brains of HD patients, but their cellular effects are not known. Here, we show that expression of microRNA-124 (miR-124) is down regulated in HD striatal mutant STHdh(Q111)/Hdh(Q111) cells, a cell model of HD compared to STHdh(Q7)/Hdh(Q7) cells. STHdh(Q7)/Hdh(Q7) and STHdh(Q111)/Hdh(Q111) cells express endogenously full length wild type and mutant HTT respectively. We confirmed this result in R6/2 mouse, an animal model of HD, expressing mutant HTT. Gene Ontology terms related to cell cycle were enriched significantly with experimentally validated targets of miR-124. We observed that expression of Cyclin A2 (CCNA2), a putative target of miR-124 was increased in mutant STHdh(Q111)/Hdh(Q111) cells and brains of R6/2 mice. Fraction of cells in S phase was higher in asynchronously growing mutant STHdh(Q111)/Hdh(Q111) cells compared to wild type STHdh(Q7)/Hdh(Q7) cells and could be altered by exogenous expression or inhibition of miR-124. Exogenous expression or knock down of CCNA2, a target of miR-124, also alters proportion of cells in S phase of HD cell model. In summary, decreased miR-124 expression could increase CCNA2 in cell and animal model of HD and is involved in deregulation of cell cycle in STHdh(Q111)/Hdh(Q111) cells.

Regulation of miR-146a by RelA/NFkB and p53 in STHdh(Q111)/Hdh(Q111) cells, a cell model of Huntington's disease.

Huntington's disease (HD) is caused by the expansion of N-terminal polymorphic poly Q stretch of the protein huntingtin (HTT). Deregulated microRNAs and loss of function of transcription factors recruited to mutant HTT aggregates could cause characteristic transcriptional deregulation associated with HD. We observed earlier that expressions of miR-125b, miR-146a and miR-150 are decreased in STHdh(Q111)/Hdh(Q111) cells, a model for HD in comparison to those of wild type STHdh(Q7)/Hdh(Q7) cells. In the present manuscript, we show by luciferase reporter assays and real time PCR that decreased miR-146a expression in STHdh(Q111)/Hdh(Q111) cells is due to decreased expression and activity of p65 subunit of NFkB (RelA/NFkB). By reporter luciferase assay, RT-PCR and western blot analysis, we also show that both miR-150 and miR-125b target p53. This partially explains the up regulation of p53 observed in HD. Elevated p53 interacts with RelA/NFkB, reduces its expression and activity and decreases the expression of miR-146a, while knocking down p53 increases RelA/NFkB and miR-146a expressions. We also demonstrate that expression of p53 is increased and levels of RelA/NFkB, miR-146a, miR-150 and miR-125b are decreased in striatum of R6/2 mice, a mouse model of HD and in cell models of HD. In a cell model, this effect could be reversed by exogenous expression of chaperone like proteins HYPK and Hsp70. We conclude that (i) miR-125b and miR-150 target p53, which in turn regulates RelA/NFkB and miR-146a expressions; (ii) reduced miR-125b and miR-150 expressions, increased p53 level and decreased RelA/NFkB and miR-146a expressions originate from mutant HTT (iii) p53 directly or indirectly regulates the expression of miR-146a. Our observation of interplay between transcription factors and miRNAs using HD cell model provides an important platform upon which further work is to be done to establish if such regulation plays any role in HD pathogenesis.

Altered microRNAs in STHdh(Q111)/Hdh(Q111) cells: miR-146a targets TBP.

We studied expression of 90 miRNAs in STHdh(Q111)/Hdh(Q111) cells, a model for Huntington's disease and compared with that obtained in STHdh(Q7)/Hdh(Q7) cells. Fifteen miRNAs were down regulated and 12 miRNAs were up regulated more than 2-fold. Such changes were statistically significant. One hundred and forty-two genes are experimentally known targets of these altered miRNAs. It has been predicted that miR-146a may target Tata Binding Protein (TBP). Using luciferase reporter assays with 3'-UTRs of TBP, over-expression and inhibition of miR-146a, we showed that miR-146a targets TBP. Regulation of TBP by miR-146a may contribute to HD pathogenesis.