JunD accentuates arecoline-induced disruption of tight junctions and promotes epithelial-to-mesenchymal transition by association with NEAT1 lncRNA.

Head and neck cancers are highly prevalent in south-east Asia, primarily due to betel nut chewing. Arecoline, the primary alkaloid is highly carcinogenic; however its role in promoting tumorigenesis by disrupting junctional complexes and increasing risk of metastasis is not well delineated. Subsequently, the effects of low and high concentrations of arecoline on the stability of tight junctions and EMT induction were studied. A microarray analysis confirmed involvement of a MAPK component, JunD, in regulating tight junction-associated genes, specifically ZO-1. Results established that although arecoline-induced phosphorylation of JunD downregulated expression of ZO-1, JunD itself was modulated by the lncRNA-NEAT1 in presence of arecoline. Increased NEAT1 in tissues of HNSCC patients significantly correlated with poor disease prognosis. Here we show that NEAT1-JunD complex interacted with ZO-1 promoter in the nuclear compartment, downregulated expression of ZO-1 and destabilized tight junction assembly. Consequently, silencing NEAT1 in arecoline-exposed cells not only downregulated the expression of JunD and stabilized expression of ZO-1, but also reduced expression of the EMT markers, Slug and Snail, indicating its direct regulatory role in arecoline-mediated TJ disruption and disease progression.

Increase in MEG3, MALAT1, NEAT1 significantly predicts the clinical parameters in patients with rheumatoid arthritis.

Aim: This study investigated deregulation of lncRNAs MEG3, MALAT1, NEAT1 and their associations with clinical parameters in rheumatoid arthritis (RA). Materials & methods: LncRNAs MALAT1, MEG3, NEAT1 were quantified from peripheral blood mono-nuclear cells (PBMCs) and plasma of 82 RA patients with 15 matched controls and from knee fluid of 24 RA patients with ten osteoarthritis controls. Multivariate analyses were performed among lncRNAs and clinical parameters of RA. Results:MALAT1, MEG3, NEAT1 were increased in PBMCs, plasma, synovial fluid (p < 0.05) of RA patients. Significant correlations were observed for MEG3 with TJC (r = 0.29), NEAT1 with TJC (r = 0.49), swollen joint count (r = 0.20), DAS28-CRP (r = 0.29). Multivariate analysis revealed that 48.5% of TJC and 31.5% of swollen joint count could be predicted by lncRNAs. Conclusion: The findings suggested that the lncRNAs might be explored as probable markers in monitoring disease activity.

E2F1 activates MFN2 expression by binding to the promoter and decreases mitochondrial fission and mitophagy in HeLa cells.

Mitofusin-2 (MFN2) is primarily involved in mitochondrial fusion and participates in diverse biological processes. Several reports show that MFN2 is a target of different miRNAs; however, the transcriptional regulation of MFN2 has not been extensively studied. To gain insight into the transcriptional regulation of MFN2, we expressed E2F transcription factor 1 (E2F1) exogenously and observed that it increased the endogenous expression of MFN2 by binding to its putative promoter region. Although the levels of E2F1 were shown to vary during the cell cycle, the expression of MFN2 and its regulator SP1 did not change throughout the different phases, suggesting that E2F1 regulates MFN2 in a cell-cycle-independent manner. In the cell-cycle phases, where the expression of E2F1 was reduced, SP1 might act in its place to regulate the expression of MFN2. We showed that E2F1 and SP1 are present as a complex on the promoter of MFN2 during the S-phase as well as in E2F1 overexpressing cells, suggesting that they may regulate the expression of MFN2 synergistically. Furthermore, we found that E2F1 modulated mitochondrial fusion and mitophagy, likely through regulation of MFN2. Bioinformatic analysis revealed that several potential targets of E2F1 are localized in mitochondria and associated with autophagy. Collectively, these data identify the E2F1-MFN2 axis as a regulator of mitochondrial morphology and mitophagy, suggesting a potential therapeutic target for the treatment of mitochondrial disorders.

Huntingtin interacting protein HYPK is a negative regulator of heat shock response and is downregulated in models of Huntington's Disease.

Huntingtin interacting protein HYPK (Huntingtin Yeast Partner K) is an intrinsically unstructured protein having chaperone-like activity and can suppress mutant huntingtin aggregates and toxicity in cell model of Huntington's Disease (HD). Heat shock response is an adaptive mechanism of cells characterized by upregulation of heat shock proteins by heat-induced activation of heat shock factor 1 (HSF1). The trans-activation ability of HSF1 is arrested upon restoration of proteostasis. We earlier identified HYPK as a heat-inducible protein and transcriptional target of HSF1. Here we show that HYPK can act as negative regulator of heat shock response by repressing transcriptional activity of HSF1. As part of its role as a repressor of heat shock response, HYPK can also inhibit HSF1-dependent trans-activation of its own promoter. HYPK is downregulated in cell and animal model of HD. We further show that transcriptional downregulation of HYPK in HD cell model is a consequence of reduced occupancy of HSF1 in HYPK promoter. Moreover, presence of mutant huntingtin inhibits effective induction of HYPK in response to heat shock. Taken together, our findings reveal that HYPK can suppress heat shock response via an autoregulatory loop and downregulation of HYPK in HD is caused by impaired transcriptional activity of HSF1 in presence of mutant huntingtin.

Trans-activation of small EDRK-rich factor 2 (SERF2) promoter by Heat Shock Factor 1.

Heat shock response is an adaptive mechanism of cells characterized by rapid synthesis of a class of proteins popularly known as heat shock proteins (HSPs) by heat-induced activation of Heat Shock Factor 1 (HSF1). In course of our earlier study to show that HSF1 regulates transcription of HYPK (Huntingtin Yeast two-hybrid protein K), a chaperone-like protein, we observed presence of few other genes within 10 kb of HYPK promoter. In an attempt to understand whether adjacent genes of HYPK are co-regulated, we identified that SERF2 (small EDRK-rich factor 2), an upstream neighboring gene of HYPK, is also regulated by heat stress and HSF1. We also showed that SERF2 promoter can be trans-activated by HSF1 due to the presence of functional heat shock element (HSE). Strikingly, HYPK is linked with SERF2 through a Conjoined Gene (CG) albeit the respective proteins have opposite effect on mutant Huntingtin aggregates and subsequent toxicity. Our study provides the first report on regulation of SERF2 expression and thereby depicts a paradigm where two parent genes of a CG are regulated by a common transcription factor despite the fact that they code for proteins having opposite cellular function in a given context.

Heat Shock Factor 1-Regulated miRNAs Can Target Huntingtin and Suppress Aggregates of Mutant Huntingtin.

BACKGROUND: Heat shock factor 1 (HSF1) is the master regulator of chaperone network in mammalian cells and can protect cells from adverse effects of misfolded proteins by rapidly inducing expression of multiple heat shock proteins (HSPs) and other cytoprotective proteins. HSF1 also regulates transcription of microRNAs (miRNAs) in heat shock-dependent manner and these miRNAs are likely to regulate diverse cellular processes by acting as downstream effectors of HSF1. METHODS: The study was aimed at understanding the effect of HSF1-regulated miRNAs on huntingtin expression and Huntington's Disease (HD) pathogenesis, if any. The cumulative effect of all HSF1-regulated miRNAs on huntingtin expression was measured by quantitative real-time PCR and luciferase reporter assay and effect of miRNAs on mutant huntingtin aggregates was determined by aggregate counting assay. RESULTS: Our study reveals that HSF1-regulated miRNAs cumulatively target huntingtin and reduce its expression in HD cell model. We also identify 4 huntingtin-targeting miRNAs viz. miR-125b, miR-146a, miR-150 and miR-214 as candidate miRNAs responsible for observed inhibitory effect of HSF1 on huntingtin expression. We further demonstrate that HSF1-regulated miRNAs together can suppress aggregates of mutant huntingtin in cell model of HD. CONCLUSION: We conclude that the protective effect of HSF1 in the context of HD is a consequence of synergistic induction of HSPs and HSF1-regulated huntingtin-targeting miRNAs. Moreover, the suppressive effect of HSF1-regulated miRNAs on mutant huntingtin aggregates indicates their potential as therapeutic agents for the treatment of HD.

Regulation of mitochondrial morphology and cell cycle by microRNA-214 targeting Mitofusin2.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the increase in CAG repeats beyond 36 at the exon1 of the gene Huntingtin (HTT). Among the various dysfunctions of biological processes in HD, transcription deregulation due to abnormalities in actions of transcription factors has been considered to be one of the important pathological conditions. In addition, deregulation of microRNA (miRNA) expression has been described in HD. Earlier, expression of microRNA-214 (miR-214) has been shown to increase in HD cell models and target HTT gene; the expression of the later being inversely correlated to that of miR-214. In the present communication, we observed that the expressions of several HTT co-expressed genes are modulated by exogenous expression of miR-214 or by its mutant. Among several HTT co-expressed genes, MFN2 was shown to be the direct target of miR-214. Exogenous expression of miR-214, repressed the expression of MFN2, increased the distribution of fragmented mitochondria and altered the distribution of cells in different phases of cell cycle. In summary, we have shown that increased expression of miR-214 observed in HD cell model could target MFN2, altered mitochondrial morphology and deregulated cell cycle. Inhibition of miR-214 could be a possible target of intervention in HD pathogenesis.

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.

Heat shock factor 1 regulates hsa-miR-432 expression in human cervical cancer cell line.

Heat shock response pathway is a conserved defense mechanism of mammalian cells to maintain protein homeostasis against proteotoxic environmental conditions. This is characterized by robust synthesis of molecular chaperones mostly by stress-induced activation of heat shock factor 1 (HSF1). MicroRNAs (miRNAs) are a family of small non-coding RNAs that negatively regulate expression of protein-coding genes. Here we report altered expression of a set of miRNAs by thermal stress in HeLa cells. We also show that HSF1 regulates hsa-miR-432 expression in heat shock-dependent manner through its cognate binding site present in hsa-miR-432 upstream sequence. Our report uncovers a novel function of HSF1 and indicates involvement of miRNAs in HSF1-mediated protection of cellular proteome.

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.

Transcription regulation of HYPK by Heat Shock Factor 1.

HYPK (Huntingtin Yeast Partner K) was originally identified by yeast two-hybrid assay as an interactor of Huntingtin, the protein mutated in Huntington's disease. HYPK was characterized earlier as an intrinsically unstructured protein having chaperone-like activity in vitro and in vivo. HYPK has the ability of reducing rate of aggregate formation and subsequent toxicity caused by mutant Huntingtin. Further investigation revealed that HYPK is involved in diverse cellular processes and required for normal functioning of cells. In this study we observed that hyperthermia increases HYPK expression in human and mouse cells in culture. Expression of exogenous Heat Shock Factor 1 (HSF1), upon heat treatment could induce HYPK expression, whereas HSF1 knockdown reduced endogenous as well as heat-induced HYPK expression. Putative HSF1-binding site present in the promoter of human HYPK gene was identified and validated by reporter assay. Chromatin immunoprecipitation revealed in vivo interaction of HSF1 and RNA polymerase II with HYPK promoter sequence. Additionally, acetylation of histone H4, a known epigenetic marker of inducible HSF1 binding, was observed in response to heat shock in HYPK gene promoter. Overexpression of HYPK inhibited cells from lethal heat-induced death whereas knockdown of HYPK made the cells susceptible to lethal heat shock-induced death. Apart from elevated temperature, HYPK was also upregulated by hypoxia and proteasome inhibition, two other forms of cellular stress. We concluded that chaperone-like protein HYPK is induced by cellular stress and under transcriptional regulation of HSF1.

Grb2 is regulated by foxd3 and has roles in preventing accumulation and aggregation of mutant huntingtin.

Growth factor receptor protein binding protein 2 (Grb2) is known to be associated with intracellular growth and proliferation related signaling cascades. Huntingtin (Htt), a ubiquitously expressed protein, when mutated, forms toxic intracellular aggregates - the hallmark of Huntington's disease (HD). We observed an elevated expression of Grb2 in neuronal cells in animal and cell models of HD. Grb2 overexpression was predominantly regulated by the transcription factor Forkhead Box D3 (Foxd3). Exogenous expression of Grb2 also reduced aggregation of mutant Htt in Neuro2A cells. Grb2 is also known to interact with Htt, depending on epidermal growth factor receptor (EGFR) activation. Grb2- mutant Htt interaction in the contrary, took place in vesicular structures, independent of EGFR activation that eventually merged with autophagosomes and activated the autophagy machinery helping in autophagosome and lysosome fusion. Grb2, with its emerging dual role, holds promise for a survival mechanism for HD.

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.