Neuron-restrictive Silencer Factor Research Articles

Possible mechanisms of lycopene amelioration of learning and memory impairment in rats with vascular dementia.

Oxidative stress is involved in the pathogenesis of vascular dementia. Studies have shown that lycopene can significantly inhibit oxidative stress; therefore, we hypothesized that lycopene can reduce the level of oxidative stress in vascular dementia. A vascular dementia model was established by permanent bilateral ligation of common carotid arteries. The dosage groups were treated with lycopene (50, 100 and 200 mg/kg) every other day for 2 months. Rats without bilateral carotid artery ligation were prepared as a sham group. To test the ability of learning and memory, the Morris water maze was used to detect the average escape latency and the change of search strategy. Hematoxylin-eosin staining was used to observe changes of hippocampal neurons. The levels of oxidative stress factors, superoxide dismutase and malondialdehyde, were measured in the hippocampus by biochemical detection. The levels of reactive oxygen species in the hippocampus were observed by dihydroethidium staining. The distribution and expression of oxidative stress related protein, neuron-restrictive silencer factor, in hippocampal neurons were detected by immunofluorescence histochemistry and western blot assays. After 2 months of drug administration, (1) in the model group, the average escape latency was longer than that of the sham group, and the proportion of straight and tend tactics was lower than that of the sham group, and the hippocampal neurons were irregularly arranged and the cytoplasm was hyperchromatic. (2) The levels of reactive oxygen species and malondialdehyde in the hippocampus of the model group rats were increased, and the activity of superoxide dismutase was decreased. (3) Lycopene (50, 100 and 200 mg/kg) intervention improved the above changes, and the lycopene 100 mg/kg group showed the most significant improvement effect. (4) Neuron-restrictive silencer factor expression in the hippocampus was lower in the sham group and the lycopene 100 mg/kg group than in the model group. (5) The above data indicate that lycopene 100 mg/kg could protect against the learning-memory ability impairment of vascular dementia rats. The protective mechanism was achieved by inhibiting oxidative stress in the hippocampus. The experiment was approved by the Animal Ethics Committee of Fujian Medical University, China (approval No. 2014-025) in June 2014.

Molecular profiling stratifies diverse phenotypes of treatment-refractory metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with diverse drivers of disease progression and mechanisms of therapeutic resistance. We conducted deep phenotypic characterization of CRPC metastases and patient-derived xenograft (PDX) lines using whole genome RNA sequencing, gene set enrichment analysis and immunohistochemistry. Our analyses revealed five mCRPC phenotypes based on the expression of well-characterized androgen receptor (AR) or neuroendocrine (NE) genes: (i) AR-high tumors (ARPC), (ii) AR-low tumors (ARLPC), (iii) amphicrine tumors composed of cells co-expressing AR and NE genes (AMPC), (iv) double-negative tumors (i.e. AR-/NE-; DNPC) and (v) tumors with small cell or NE gene expression without AR activity (SCNPC). RE1-silencing transcription factor (REST) activity, which suppresses NE gene expression, was lost in AMPC and SCNPC PDX models. However, knockdown of REST in cell lines revealed that attenuated REST activity drives the AMPC phenotype but is not sufficient for SCNPC conversion. We also identified a subtype of DNPC tumors with squamous differentiation and generated an encompassing 26-gene transcriptional signature that distinguished the five mCRPC phenotypes. Together, our data highlight the central role of AR and REST in classifying treatment-resistant mCRPC phenotypes. These molecular classifications could potentially guide future therapeutic studies and clinical trial design.

RE-1 silencing transcription factor alleviates the growth-suppressive effects of propofol on mouse neuronal cells.

Propofol is broadly utilized for maintaining anesthesia. Propofol affects neurodegeneration and neurogenesis by regulation of autophagy via effects on intracellular calcium homeostasis. The underlying molecular mechanism, however, is still unclear. In the present research, we systematically analyzed the effect of propofol on mouse neuronal cells (cell line: HT-22). Cell Counting Kit-8 assays were utilized to examine cell proliferation. Flow cytometry was used to determine the levels of cell apoptosis. Quantitative real-time PCR and western blot were used to examine the relative mRNA and protein levels in mouse neuronal cells. Our results suggest that propofol inhibits proliferation and promotes apoptosis in neuronal cells. Moreover, overexpression of the transcriptional repressor RE-1 silencing transcription factor rescued the effect of propofol on neuronal cells. Additionally, the autophagy inhibitor 3-methyladenine also significantly reduced the effect of propofol on mouse neuronal cells. Finally, overexpression of RE-1 silencing transcription factor promoted the expression of brain-derived neurotrophic factor in mouse neuronal cells. Our research not only enhances our understanding of propofol on mouse neuronal cells but also uncovers a potential signaling pathway that may mediate the effects of propofol on neuronal cells.

Long Non-coding RNA ITIH4-AS1 Accelerates the Proliferation and Metastasis of Colorectal Cancer by Activating JAK/STAT3 Signaling

Accumulating evidence has uncovered long non-coding RNAs (lncRNAs) as central regulators in the pathogenesis of diverse human cancers including colorectal cancer (CRC). The present study discovered that a novel lncRNA ITIH4 antisense RNA 1 (ITHI4-AS1) was frequently under-expressed in most normal human tissues, including colon tissues. Therefore, we aimed to investigate the role of ITHI4-AS1 in CRC. Interestingly, a significant overexpression of ITIH4-AS1 was observed in CRC cell lines relative to normal NCM460 cells. Also, we investigated the facilitating role of ITIH4-AS1 in CRC cell growth and metastasis both in vitro and in vivo. Additionally, we explained that ITIH4-AS1 upregulation in CRC was attributed to downregulation or even depletion of RE1 silencing transcription factor (REST), a presently identified transcriptional repressor for ITIH4-AS1. Meanwhile, the contribution of ITIH4-AS1 to CRC development was validated to rely on the activation of the JAK/STAT3 pathway. More importantly, we verified that FUS interacted with both ITIH4-AS1 and STAT3, and that ITIH4-AS1 evoked nuclear translocation of phosphorylated (p)-STAT3 in CRC through recruiting FUS. In summary, our findings unveiled for the first time that REST downregulation-enhanced ITIH4-AS1 exerts pro-tumor functions in CRC through FUS-dependent activation of the JAK/STAT3 pathway, implying that targeting ITIH4-AS1 may be a novel effective strategy for CRC therapy.

Commensal Bacteria Regulate Gene Expression and Differentiation in Vertebrate Olfactory Systems Through Transcription Factor REST.

Sensory systems such as the olfactory system detect chemical stimuli and thereby determine the relationships between the animal and its surroundings. Olfaction is one of the most conserved and ancient sensory systems in vertebrates. The vertebrate olfactory epithelium is colonized by complex microbial communities, but microbial contribution to host olfactory gene expression remains unknown. In this study, we show that colonization of germ-free zebrafish and mice with microbiota leads to widespread transcriptional responses in olfactory organs as measured in bulk tissue transcriptomics and RT-qPCR. Germ-free zebrafish olfactory epithelium showed defects in pseudostratification; however, the size of the olfactory pit and the length of the cilia were not different from that of colonized zebrafish. One of the mechanisms by which microbiota control host transcriptional programs is by differential expression and activity of specific transcription factors (TFs). REST (RE1 silencing transcription factor, also called NRSF) is a zinc finger TF that binds to the conserved motif repressor element 1 found in the promoter regions of many neuronal genes with functions in neuronal development and differentiation. Colonized zebrafish and mice showed increased nasal expression of REST, and genes with reduced expression in colonized animals were strongly enriched in REST-binding motifs. Nasal commensal bacteria promoted in vitro differentiation of Odora cells by regulating the kinetics of REST expression. REST knockdown resulted in decreased Odora cell differentiation in vitro. Our results identify a conserved mechanism by which microbiota regulate vertebrate olfactory transcriptional programs and reveal a new role for REST in sensory organs.

Young Blood Rescues the Cognition of Alzheimer's Model Mice by Restoring the Hippocampal Cholinergic Circuit

An increasing number of studies have demonstrated the benefits of young individual-derived blood for aging-related diseases. However, the effects of young blood on the cognitive and cholinergic transmission defects in aging-associated Alzheimer's disease (AD) remain elusive. In the current study, we showed that young blood serum delivered intravenously attenuated deficits in hippocampal-dependent learning and memory, alleviated hippocampal Aβ plaque pathology, restored synapse formation and synaptic plasticity, repaired the hippocampal cholinergic circuit, and triggered several canonical neuroprotective mechanisms [including repressor element 1-silencing transcription factor (REST)/Forkhead box protein O1 (FOXO1) signaling] in aged AD model mice. However, pharmacological blockage of hippocampal cholinergic activity nearly abrogated the neuroprotective actions of young blood serum in AD mice. Thus, our findings suggest that exogenous young blood serum exerts therapeutic effects on AD-associated cognitive disorders and pathology by promoting hippocampal cholinergic input and simultaneously activating other neuroprotective mechanisms.

Abstract 652: Increased Gα o Expression Plays a Pivotal Role in the Progression of Heart Failure by Impairing Ca 2+ Handling

Background: A transcriptional repressor, neuron restrictive silencer factor (NRSF), maintains normal cardiac function and electrical stability. Transgenic mice expressing a dominant-negative mutant of NRSF in their hearts (dnNRSF-Tg) exhibit systolic dysfunction and premature death due to lethal arrhythmias like human dilated cardiomyopathy(DCM). Underlining mechanisms remain to be elucidated, however. Purpose: To clarify how NRSF maintains normal cardiac homeostasis. Methods and Results: cDNA microarray analysis of dnNRSF-Tg and newly generated cardiac-specific NRSF knockout mice (NRSFcKO), which show similar cardiac phenotypes to those of dnNRSF-Tg, revealed that cardiac gene expression of GNAO1 that encodes Gα o , a member of inhibitory G protein Gα i family, is increased in both dnNRSF-Tg and NRSFcKO ventricles. ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay identified that GNAO1 is a direct target of NRSF. In dnNRSF-Tg, pharmacological inhibition of Gα o by pertussis toxin improved systolic dysfunction and knockdown of Gα o by crossing with GNAO1 knockout mice improved not only systolic function but also frequency of ventricular arrhythmias and survival rates. Electrophysiological analysis in ventricular myocytes obtained from dnNRSF-Tg demonstrated that genetic reduction of Gα o ameliorated abnormalities in Ca 2 + handling, which include increased current density in surface sarcolemmal L-type Ca 2 + channel, reduced content of sarcoplasmic reticulum Ca 2+ and lowered peak of Ca 2+ transient. In addition, genetic reduction of Gα o attenuated increased phosphorylation levels of CAMKII and RyR2 in dnNRSF-Tg ventricles, which presumably underlies the improvement in Ca 2 + handling. Furthermore, we identified increased Gα o expression in ventricles of heart failure model mice induced by transverse aortic constriction and cardiac troponin T mutant DCM model mice, in both of which, genetic reduction of Gα o ameliorated cardiac dysfunction. Conclusions: We found that increased expression of Gα o , induced by attenuation of NRSF-mediated repression, plays a pivotal role in the progression of heart failure by evoking Ca 2+ handling abnormality. Gα o is a potential therapeutic target for heart failure.

CoRest1 regulates neurogenesis in a stage-dependent manner.

Developmental processes, including neuronal differentiation, require precise regulation of transcription. The RE-1 silencing transcription factor (Rest), is often called a "master neuronal regulator" due to its large number of neural-specific targets. Rest recruits CoRest (Rcor) and Sin3 corepressor complexes to gene regulatory sequences. CoRest not only associates with Rest, but with other transcription regulators. In this study, we generated zebrafish rcor1 mutants using transcription activator-like effector nucleases (TALENS), to study its requisite role in repression of Rest target genes as well as Rest-independent Rcor1 developmental functions. While rcor1 mutants have a slight decrease in fitness, most survived and produced viable offspring. We examined expression levels of RE1-containing genes in maternal zygotic rcor1 (MZrcor1) mutants and found that Rcor1 is generally not required for the repression of Rest target genes at early stages. However, MZrcor1 mutants undergo more rapid neurogenesis compared to controls. We found that at gastrula stages, Rcor1 acts as a repressor of her gene family, but at later stages, her6 decreased in the MZrcor1 mutant. Based on these findings, the central role of CoRest1 in neurogenesis is likely due to a Rest-independent role rather than as a Rest corepressor.

The interaction between RE1-silencing transcription factor (REST) and heat shock protein 90 as new therapeutic target against Huntington's disease.

The wild type huntingtin protein (Htt), supports the production of brain-derived neurotrophic factor (BDNF), a survival factor for striatal neurons, through cytoplasmic sequestering of RE-1silencing transcription factor (REST). In Huntington´s Disease an inherited degenerative disease, caused by a CAG expansion in the 5´coding region of the gene, the mutant huntingtin protein (mHtt), causes that REST enters pathologically into the nucleus of cells, resulting in the repression of neuronal genes including BDNF, resulting in the progressive neuronal death. It has been reported that Htt associates with Hsp90 and this interaction is involved in regulation of huntingtin aggregation. Discovering mechanisms to reduce the cellular levels of mutant huntingtin and REST provide promising strategies for treating Huntington disease. Here, we use the yeast two-hybrid system to show that N-terminus or REST interacts with the heat shock protein 90 (Hsp90) and identifies REST as an Hsp90 Client Protein. To assess the effects of Hsp90 we used antisense oligonucleotide, and evaluated the levels mHtt and REST levels. Our results show that direct knockdown of endogenous Hsp90 significantly reduces the levels of REST and mutant Huntingtin, decreased the percentage of cells with mHtt in nucleus and rescued cells from mHtt-induced cellular cytotoxicity. Additionally Hsp90–specific inhibitors geldanamicyn and PUH71 dramatically reduced mHtt and REST levels, thereby providing neuroprotective activity. Our data show that Hsp90 is necessary to maintain the levels of REST and mHtt, which suggests that the interactions between Hsp90-REST and Hsp90-Huntingtin could be potential therapeutic targets in Huntington's disease.

Loss of Cln5 leads to altered Gad1 expression and deficits in interneuron development in mice.

The Finnish-variant late infantile neuronal ceroid lipofuscinosis, also known as CLN5 disease, is caused by mutations in the CLN5 gene. Cln5 is strongly expressed in the developing brain and expression continues into adulthood. CLN5, a protein of unknown function, is implicated in neurodevelopment but detailed investigation is lacking. Using Cln5-/- embryos of various ages and cells harvested from Cln5-/- brains we investigated the hitherto unknown role of Cln5 in the developing brain. Loss of Cln5 results in neuronal differentiation deficits and delays in interneuron development during in utero period. Specifically, the radial thickness of dorsal telencephalon was significantly decreased in Cln5-/- mouse embryos at embryonic day 14.5 (E14.5), and expression of Tuj1, an important neuronal marker during development, was down-regulated. An interneuron marker calbindin and a mitosis marker p-H3 showed down-regulation in ganglionic eminences. Neurite outgrowth was compromised in primary cortical neuronal cultures derived from E16 Cln5-/- embryos compared with WT embryos. We show that the developmental deficits of interneurons may be linked to increased levels of the repressor element 1-silencing transcription factor, which we report to bind to glutamate decarboxylase (Gad1), which encodes GAD67, a rate-limiting enzyme in the production of gamma-aminobutyric acid (GABA). Indeed, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons. Furthermore, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons and showed age-independent cortical hyper excitability as measured by electroencephalogram and auditory-evoked potentials. This study highlights the importance of Cln5 in neurodevelopment and suggests that in contrast to earlier reports, CLN5 disease is likely to develop during embryonic stages.

High Aβ load may cause microglial cell dysfunction and reduced nuclear repressor element-1 silencing transcription factor (REST) expression which might be ascribed to its degradation by ubiquitination.

This study aimed to investigate the effects of high Aβ load on the microglial function, neuronal activity, and repressor element-1 silencing transcription factor (REST) expression and nuclear localization and further explore the mechanism underlying nuclear REST deficiency and the correlation between pathology and symptoms in Alzheimer's disease (AD). BV2 cells and N2A cells were treated with Aβ at different concentrations, and the viability, apoptosis and autophagy of these cells were further evaluated. The expression of major histocompatibility complex class II (MHC-II, a marker of microglial activation), REST in the neurons and REST specific phosphorylase casein kinase 1 (CK1) were detected. Aβ at a low concentration (2.5 µmol/L) could alter the microglial morphology, and the proportion of amoebic microglia and protein expression of MHC-II increased in a Aβ concentration dependent manner. Aβ at a high concentration (10 µmol/L) was able to reduce REST expression, elevate the expression of pro-apoptotic and pro-autophagic genes, inhibit the expression of anti-apoptotic gene, and reduce the neuronal activity. REST expression reduced, but CK1 increased in neurons, and CK1 inhibitor significantly increased REST expression. there was co-expression of REST and CK1 in the brain of AD mice, which was characterized by reduced nuclear REST expression and elevated CK1 expression. High Aβ load may cause microglial cell dysfunction and loss of REST expression in the neurons, resulting in dementia. The reduced nuclear REST might be ascribed to its degradation by ubiquitination. This supports the hypothesis that high plaque load may increase the risk for dementia.

Abstract 4392: Pre-mRNA splicing factors promote cellular plasticity in castration-resistant prostate cancer

Abstract Background: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with poorly understood drivers of disease progression. We recently characterized five mCRPC phenotypes, including an amphicrine phenotype that co-expresses androgen receptor (AR) and neuroendocrine prostate cancer (NEPC) biomarkers. We determined that loss of RE1-silencing transcription factor (REST), a master regulator of neuronal differentiation, drives the amphicrine phenotype, but is not sufficient for prostate cancer (PC) adenocarcinoma to NEPC conversion. Furthermore, loss of REST through SRRM4-mediated splicing of REST pre-mRNA to REST4 has been suggested to drive adenocarcinoma to NEPC conversion. However, the roles of pre-mRNA splicing factors and REST activity in lineage switching require further investigation. Methods: Transcriptomic (RNASeq) and immunohistochemical/immunofluorescent analysis (IHC and IF) were conducted on amphicrine and NEPC patient metastases, LuCaP patient-derived xenograft (PDX) models and modified CRPC cell lines. The roles of SRRM3 and SRRM4 were examined using overexpression studies in AR-expressing and AR-null CRPC cell lines. Results: RNASeq, IHC and IF of metastatic specimens, LuCaP PDX models and VCaP cells confirmed the existence of the amphicrine phenotype in vitro and in vivo. Interestingly, transcriptome analysis of amphicrine patient specimens and LuCaP 77CR revealed that loss of REST repressor activity occurred without SRRM4 expression in a subset of tumor specimens. Indeed, BaseScope analysis using primers specific to REST4 and SRRM4 verified that amphicrine 77CR tumors were positive for REST4 expression but negative for SRRM4 expression, suggesting an alternative mechanism of REST splicing. Notably, overexpression of SRRM4 in AR-expressing C4-2B and AR-null PC-3 cells did not induce REST splicing. Moreover, RNASeq of SRRM4-overexpressing cells displayed heterogeneous transcriptome profiles inconsistent with canonical amphicrine or NEPC gene expression profiles. Interestingly, SRRM3 transcript was expressed at high levels in amphicrine and NEPC patient and LuCaP PDX biospecimens that lacked SRRM4 expression, suggesting an SRRM3-mediated mechanism of REST splicing. Studies interrogating the roles of SRRM3 in REST splicing and CRPC cellular plasticity are ongoing. Conclusions: Our data highlights an unrecognized mechanism of adenocarcinoma to amphicrine or NEPC conversion that hinges on a SRRM3-REST regulatory axis rather than REST-loss or SRRM4-mediated REST splicing. Identifying the mechanisms that may convert adenocarcinoma to treatment-resistant amphicrine or NEPC phenotypes in mCRPC patients will inform treatment and identify potential molecular pathways for therapeutic intervention. Citation Format: Mark P. Labrecque, Ilsa M. Coleman, Lisha G. Brown, Bryce Lakely, Lori Kollath, Daniel W. Lin, Lawrence D. True, Eva Corey, Peter S. Nelson, Colm Morrissey. Pre-mRNA splicing factors promote cellular plasticity in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4392.

Abstract 5225: Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

Abstract The Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) overexpression represents the second-largest prostate cancer (PCa) subtype associated with increased risk of recurrence and poor prognosis. Regardless of molecular subtype, androgen-deprivation therapy (ADT) remains the mainstay treatment for locally advanced and metastatic PCa patients. However, majority of the treated individuals eventually progress to castration-resistant stage and a subset of these patients develop ADT-induced neuroendocrine prostate cancer. Despite evidences of detrimental effects of ADT on PCa, possible role of androgen signaling in SPINK1-mediated prostate oncogenesis remains unexplored. Here, we show that androgen receptor (AR) functions as a direct transcriptional repressor of SPINK1, and blocking AR signaling relieves its repression, leading to upregulation of SPINK1. In agreement, we observe an inverse association between SPINK1 levels and AR expression across multiple patient cohorts, and in neuroendocrine differentiated LNCaP cells. We show that AR and its corepressor, the RE1-silencing transcription factor (REST), occupy SPINK1 promoter and inhibits its transcription. On the other hand, in the absence of AR, lineage reprogramming factor SOX2 in turn binds to SPINK1 promoter leading to its positive transcriptional regulation in androgen-deprived conditions with concomitant increase in neuroendocrine markers. Additionally, stable knockdown of SPINK1 results in reduced epithelial-mesenchymal transition, decreased stemness and drug resistance. Collectively, our findings provide a plausible explanation to the paradoxical clinical outcomes of ADT, arising due to increase in SPINK1 levels. Finally, we emphasize the need to take a well-informed decision prior to ADT and develop alternative therapeutic strategies for castrate-resistant PCa patients. Citation Format: Ritika Tiwari, Nishat Manzar, Vipul Bhatia, Anjali Yadav, Shannon Carskadon, Nilesh Gupta, Amina Zoubeidi, Nallasivam Palanisamy, Bushra Ateeq. Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5225.

Abstract 1852: Down-regulation of PRMT6 drives Warburg effect through ERK induced relocalization of PKM2 to promote tumorigenicity and sorafenib resistance in hepatocellular carcinoma

Abstract Cancer cells have a high dependence of glycolysis for ATP production, especially under hypoxic environment. Metabolic reprogramming in cancer drives an increased glycolytic rate that supports maximal production of nutrients for tumorigenesis. Our group has recently identified the post-translational modification enzyme, protein arginine methyltransferase 6 (PRMT6) to be frequently down-regulated and to exhibit a tumour suppressive effect in maintenance of cancer stemness in hepatocellular carcinoma (HCC) (Chan LH et al. Cell Reports 2018). Our current study finds transcriptome and metabolome profiling of HCC cells with PRMT6 repressed to exhibit enrichment of genes and metabolites involved in glycolysis. In vitro, PRMT6 negatively regulates glycolysis, glucose uptake, lactate production and pyruvate kinase activity in HCC cell lines and patient-derived organoids. Our previous work found PRMT6 to interfere with RAS/RAF binding by directly binding to CRAF and methylating arginine100 residue. Supporting the importance of PRMT6-mediated CRAF methylation, we find overexpression of catalytic inactive PRMT6 to exhibit no effect on glycolysis. MEK/ERK, known downstream effectors of RAS/RAF signalling, has been shown to induce nuclear PKM2 localization. Consistently, we find modulation of PRMT6 to negatively regulate ERK and PKM2 expression and localization. Rescue experiments involving the ERK inhibitor U0126 and shPKM2 further substantiate the importance of ERK/PKM2-mediated glucose metabolic reprogramming in HCC cells with PRMT6 suppressed. Functionally, inhibition of glycolysis by 2-deoxyglucose sensitized PRMT6 knockdown cells to sorafenib treatment in vitro and attenuated tumor growth in vivo. In addition, we also find PRMT6 expression to be regulated via hypoxia by binding of RE1-silencing transcription factor to the promoter region. Rescue experiments by re-expressing PRMT6 under hypoxic conditions further support the involvement of PRMT6 in driving glycolysis in oxygen deprived environments. Our work uncovers a critical function for PRMT6 in driving Warburg effect through regulating expression and localization of PKM2 via post-translational modification of CRAF, thereby promoting HCC initiation and sorafenib resistance. Citation Format: Tin-Lok Wong, Lok-Hei Chan, Stephanie Ma. Down-regulation of PRMT6 drives Warburg effect through ERK induced relocalization of PKM2 to promote tumorigenicity and sorafenib resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1852.

Repressor element 1-silencing transcription factor drives the development of chronic pain states.

Chronic pain is an unmet clinical problem with vast individual, societal, and economic impact. Pathologic activity of the peripheral somatosensory afferents is one of the major drivers of chronic pain. This overexcitable state of somatosensory neurons is, in part, produced by the dysregulation of genes controlling neuronal excitability. Despite intense research, a unifying theory behind neuropathic remodelling is lacking. Here, we show that transcriptional suppressor, repressor element 1-silencing transcription factor (REST; neuron-restrictive silencing factor, NRSF), is necessary and sufficient for the development of hyperalgesic state after chronic nerve injury or inflammation. Viral overexpression of REST in mouse dorsal root ganglion (DRG) induced prominent mechanical and thermal hyperalgesia in vivo. Sensory neuron-specific, inducible Rest knockout prevented the development of such hyperalgesic state in 3 different chronic pain models. Genetic deletion of Rest reverted injury-induced hyperalgesia. Moreover, viral overexpression of REST in the same neurons in which its gene has been genetically deleted restored neuropathic hyperalgesia. Finally, sensory neuron specific Rest knockout prevented injury-induced downregulation of REST target genes in DRG neurons. This work identified REST as a major regulator of peripheral somatosensory neuron remodelling leading to chronic pain. The findings might help to develop a novel therapeutic approache to combat chronic pain.

Production and characterization of monoclonal antibodies against the DNA binding domain of the RE1-silencing transcription factor.

The use of monoclonal antibodies for the detection of cellular biomarkers during carcinogenesis provides new strategies for cancer diagnosis or prognosis in patients. Loss of the Restrictive Element 1-Silencing Transcription (REST) factor has been observed in previous molecular and immunological approaches in aggressive breast cancer, small cell lung cancer, liver carcinoma, and colo-rectal cancer; however, for clinic diagnosis, monoclonal antibodies for REST recognition are unavailable. The goal of this work was to design, produce and characterize monoclonal antibodies against the REST DNA binding damain (DBD) that would be suitable for immunoassays. We searched for conserved domains, and immunogenic and antigenic sites in the REST structure via in silico analysis. For mice immunization, we used a recombinant REST DBD purified by affinity chromatography, and then Hybridomas were generated by mouse spleen fusion with myeloma cells. Finally, for monoclonal antibody characterization, we performed enzyme-linked immunosorbent (ELISA), western blot, dot blot, immunocytochemistry (ICC) and immunoprecipitation assays. Results showed that the DBD is conserved in REST isoforms and contains immunogenic and antigenic sites. We generated three clones producing monoclonal antibodies against REST DBD, one of them specifically recognized native REST and was suitable for ICC in samples from patients.

Brain-specific NRSF deficiency aggravates dopaminergic neurodegeneration and impairs neurogenesis in the MPTP mouse model of Parkinson's disease.

Degeneration of the dopaminergic neurons in the substantia nigra and the resultant dopamine depletion from the striatum are the hallmarks of Parkinson’s disease (PD) and are responsible for the disease’s cardinal motor symptoms. The transcriptional repressor Neuron-Restrictive Silencer Factor (NRSF), also known as RE1-Silencing Transcription Factor (REST), was originally identified as a negative regulator of neuron-specific genes in non-neuronal cells. Our previous study showed that mice deficient in neuronal NRSF/REST expression were more vulnerable to the noxious effects of the dopaminergic neurotoxin MPTP. Here, we found that brain-specific deletion of NRSF/REST led to more severe damages to the nigrostriatal pathway and long-lasting behavioral impairments in mice challenged with MPTP. Moreover, compared to wild-type controls, these mice showed increased neurogenesis shortly after MPTP exposure, but reduced neurogenesis later on. These results suggest that NRSF/REST acts as a negative modulator of neurogenesis and a pro-survival factor of neural stem cells under both normal conditions and during the course of PD.

Increased Expression of lncRNA UCA1 and HULC Is Required for Pro-inflammatory Response During LPS Induced Sepsis in Endothelial Cells.

Systemic uncontrolled inflammatory response, also termed as sepsis, is responsible for many mortalities. Bacterial endotoxin, lipopolysaccharide (LPS), is a major cause of sepsis in endothelial cells. Even though a lot of research has been done to define underlying mechanisms of LPS induced sepsis, the role of long non-coding RNAs (lncRNAs), a group of >200 kb RNAs in sepsis is not well-defined. Expression of pro-inflammatory mediators IL6, ICAM1, and VCAM1 (which encodes interleukin-6, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, respectively) were determined following LPS treatment of human dermal microvascular endothelial cells (HMECs) for 24 h to confirm sepsis induction. RNA immunoprecipitation (RIP) analysis was performed using the chromatin modifying proteins (CMPs), heterogeneous nuclear ribonucleoprotein (hnRNP) K and corepressors of the RE-1 silencing transcription factor (coREST) as individual baits. Quantitative real time polymerase chain reaction (qRT-PCR) was performed on RNA isolated from immunoprecipitated pellets for six different lncRNAs. The effect of the differentially expressed lncRNAs were determined by ectopic overexpression of the lncRNAs before induction of sepsis. Expression of IL6, ICAM1, and VCAM1 were significantly upregulated following treatment of the HMECs with LPS for 24 h confirming induction of sepsis. RIP and qRT-PCR analysis revealed that the lncRNAs HULC, UCA1, and MALAT-1 were significantly enriched with the CMPs after sepsis. RNA interference using siRNAs targeting HULC and UCA1, but not MALAT-1, decreased the expression of IL6, ICAM1, and VCAM1 to endogenous levels. Our results were further validated in an in vivo model of sub-lethal LPS-induced sepsis, whereby siRNA mediated knockdown of UCA1 and HULC lncRNAs prevented induction of VCAM1, ICAM1, and IL6, as assayed by immunohistochemistry. Cumulatively, these results suggest that LPS induced in vitro sepsis in endothelial cells and induction of pre-inflammatory mediators are at least in part due to increased expression of the UCA1 and HULC lncRNAs.

Overexpression of miR-9 in the Nucleus Accumbens Increases Oxycodone Self-Administration.

BackgroundThere is an urgent need to identify factors that increase vulnerability to opioid addiction to help stem the opioid epidemic and develop more efficient pharmacotherapeutics. MicroRNAs are small non-coding RNAs that regulate gene expression at a posttranscriptional level and have been implicated in chronic drug-taking in humans and in rodent models. Recent evidence has shown that chronic opioid treatment regulates the microRNA miR-9. The present study was designed to test the hypothesis that miR-9 in the nucleus accumbens potentiates oxycodone addictive-like behavior.MethodsWe utilized adeno-associated virus (AAV) to overexpress miR-9 in the nucleus accumbens of male rats and tested the effects on intravenous self-administration of the highly abused prescription opioid, oxycodone, in 1-hour short-access followed by 6-h long-access sessions, the latter of which leads to escalation of drug intake. In separate rats, we assessed the effects of nucleus accumbens miR-9 overexpression on mRNA targets including RE1-silencing transcription factor (REST) and dopamine D2 receptor (DRD2), which have been shown to be regulated by drugs of abuse.ResultsOverexpression of miR-9 in the nucleus accumbens significantly increased oxycodone self-administration compared with rats expressing a control, scrambled microRNA. Analysis of the pattern of oxycodone intake revealed that miR-9 overexpression increased “burst” episodes of intake and decreased the inter-infusion interval. Furthermore, miR-9 overexpression decreased the expression of REST and increased DRD2 in the nucleus accumbens at time points that coincided with behavioral effects.ConclusionsThese results suggest that nucleus accumbens miR-9 regulates oxycodone addictive-like behavior as well as the expression of genes that are involved in drug addiction.

Identification of RE1-Silencing Transcription Factor as a Promoter of Metastasis in Pancreatic Cancer.

Pancreatic cancer is characterized by its rapid progression and early metastasis. This requires further elucidation of the key promoters for its progression and metastasis. In this study, we identified REST as the hub gene of a gene module which is closely associated with cancer stage by weighted gene correlation network analysis. Validation with the TCGA database, western blot analysis of human pancreatic cancer cell lines (AsPC-1, Capan-2, SW-1990, and PANC-1) and immunohistochemical analysis of paraffin-embedded pancreatic cancer tissue sections showed that REST was enriched in tissue samples of advanced stage and metastatic phenotype cell lines. Survival analysis with the TCGA database and our own follow-up data suggested that patients with higher expression level of REST showed worse overall survival rate. In vitro functional experiments suggested that knockdown of REST suppressed proliferation, migration, invasion and epithelial-mesenchymal transition of AsPC-1 and PANC-1 cells. In vivo experiments (a subcutaneous BALB/c nude mouse model and a superior mesenteric vein injection BALB/c nude mouse model) suggested that knockdown of REST suppressed growth and metastasis of xenograft tumor. Finally, we investigated the underlying molecular mechanism of REST and identified REST as a potential downstream target of MAPK signaling pathway. In conclusion, our results of bioinformatic analysis, in vitro and in vivo functional analysis suggested that REST may serve as a promoter of metastasis in pancreatic cancer.