1 2326 133 EPIGENETIC REGULATION OF HOTAIR IN ADVANCED CHRONIC MYELOID LEUKEMIA. PURPOSE: CHRONIC MYELOID LEUKEMIA (CML) ACCOUNTS FOR ~10% OF LEUKEMIA CASES, AND ITS PROGRESSION INVOLVES EPIGENETIC GENE REGULATION. THIS STUDY INVESTIGATED EPIGENETIC REGULATION OF HOTAIR AND ITS TARGET MICRORNA, MIR-143, IN ADVANCED CML. PATIENTS AND METHODS: WE FIRST ISOLATED BONE MARROW MONONUCLEAR CELLS FROM 70 PATIENTS WITH DIFFERENT PHASES OF CML AND FROM HEALTHY DONORS AS NORMAL CONTROL; WE ALSO CULTURED K562 AND KCL22 CELLS, TREATED WITH DEMETHYLATION DRUG; MTT ASSAY, FLOW CYTOMETRY, QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), WESTERN BLOT, LUCIFERASE ASSAY, RNA PULL-DOWN ASSAY AND RNA-BINDING PROTEIN IMMUNOPRECIPITATION (RIP) ASSAY WERE PERFORMED. RESULT: AS MEASURED BY QPCR, HOTAIR EXPRESSION IN K562 CELLS, KCL22 CELLS, AND SAMPLES FROM CASES OF ADVANCED-STAGE CML INCREASED WITH LEVELS OF SEVERAL DNA METHYLTRANSFERASES AND HISTONE DEACETYLATES, INCLUDING DNMT1, DNMT3A, HDAC1, EZH2, AND LSD1, AND MIR-143 LEVELS WERE DECREASED AND HOTAIR LEVELS WERE INCREASED. TREATMENT WITH 5-AZACYTIDINE, A DNA METHYLATION INHIBITOR, DECREASED DNMT1, DNMT3A, HDAC1, EZH2, LSD1 MRNA, PROTEIN LEVELS, AND HOTAIR MRNA LEVELS BUT INCREASED MIR-143 LEVELS. HOTAIR KNOCKDOWN AND MIR-143 OVEREXPRESSION BOTH INHIBITED PROLIFERATION AND PROMOTED APOPTOSIS IN KCL22 AND K562 CELLS THROUGH THE PI3K/AKT PATHWAY. RNA PULL-DOWN, MASS SPECTROMETRY, AND RIP ASSAYS SHOWED THAT HOTAIR INTERACTED WITH EZH2 AND LSD1. A DUAL-LUCIFERASE ASSAY DEMONSTRATED THAT HOTAIR INTERACTED WITH MIR-143. CONCLUSION: OUR FINDINGS DEMONSTRATE THE KEY EPIGENETIC INTERACTIONS OF HOTAIR RELATED TO CML PROGRESSION AND SUGGEST HOTAIR AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AS A CML TREATMENT STRATEGY. 2018 2 1632 41 DNMTS ARE INVOLVED IN TGF-BETA1-INDUCED EPITHELIAL-MESENCHYMAL TRANSITIONS IN AIRWAY EPITHELIAL CELLS. CHRONIC RHINOSINUSITIS (CRS) PATHOGENESIS IS CLOSELY RELATED TO TISSUE REMODELING, INCLUDING EPITHELIAL-MESENCHYMAL TRANSITION (EMT). EPIGENETIC MECHANISMS PLAY KEY ROLES IN EMT. DNA METHYLATION, MEDIATED BY DNA METHYLTRANSFERASES (DNMTS), IS AN EPIGENETIC MARKER THAT IS CRITICAL TO EMT. THE GOAL OF THIS STUDY WAS TO DETERMINE WHETHER DNMTS WERE INVOLVED IN TGF-BETA1-INDUCED EMT AND ELUCIDATE THE UNDERLYING MECHANISMS IN NASAL EPITHELIAL CELLS AND AIR-LIQUID INTERFACE CULTURES. GLOBAL DNA METHYLATION AND DNMT ACTIVITY WERE QUANTIFIED. DNMT EXPRESSION WAS MEASURED USING REAL-TIME PCR (QRT-PCR) IN HUMAN CRS TISSUES. MRNA AND PROTEIN LEVELS OF DNMTS, E-CADHERIN, VIMENTIN, ALPHA-SMA, AND FIBRONECTIN WERE DETERMINED USING RT-PCR AND WESTERN BLOTTING, RESPECTIVELY. DNMT1, DNMT3A, AND DNMT3B GENE EXPRESSION WERE KNOCKED DOWN USING SIRNA TRANSFECTION. MAPK PHOSPHORYLATION AND EMT-RELATED TRANSCRIPTION FACTOR LEVELS WERE DETERMINED USING WESTERN BLOTTING. SIGNALING PATHWAYS WERE ANALYZED USING SPECIFIC INHIBITORS OF MAPK. WE DEMONSTRATED THESE DATA IN PRIMARY NASAL EPITHELIAL CELLS AND AIR-LIQUID INTERFACE CULTURES. GLOBAL DNA METHYLATION, DNMT ACTIVITY, AND DNMT EXPRESSION INCREASED IN CRS TISSUES. DNMT EXPRESSION WAS POSITIVELY CORRELATED WITH LUND-MCKAY CT SCORES. TGF-BETA1 DOSE-DEPENDENTLY INDUCED DNMT EXPRESSION. FURTHER, 5-AZA INHIBITED TGF-BETA1-INDUCED DNMT, SNAIL, AND SLUG EXPRESSION RELATED TO EMT, AS WELL AS P38 AND JNK PHOSPHORYLATION IN A549 CELLS AND TGF-BETA1-INDUCED DNMT EXPRESSION AND EMT IN PRIMARY NASAL EPITHELIAL CELLS AND AIR-LIQUID INTERFACE CULTURES. TGF-BETA1-INDUCED DNMT EXPRESSION LEADS TO DNA METHYLATION AND EMT VIA P38, JNK, SNAIL, AND SLUG SIGNALING PATHWAYS. INHIBITION OF DNMT SUPPRESSED THE EMT PROCESS AND THEREFORE IS POTENTIALLY A CRS THERAPEUTIC STRATEGY. 2022 3 5459 61 RESEARCH ON THE EPIGENETIC REGULATION MECHANISM OF THE PTPN6 GENE IN ADVANCED CHRONIC MYELOID LEUKAEMIA. PTPN6, A TYROSINE PHOSPHATASE PROTEIN, PLAYS A NEGATIVE ROLE IN CELL SIGNAL TRANSDUCTION AND IS NEGATIVELY CORRELATED WITH TUMOUR FORMATION AND GROWTH. HOWEVER, EPIGENETIC REGULATION MECHANISM OF THE PTPN6 GENE IN ADVANCED CHRONIC MYELOID LEUKAEMIA (CML) REMAINS UNCLEAR. THIS STUDY INVESTIGATED BONE MARROW OR BLOOD SAMPLES FROM 44 CML PATIENTS AND 10 HEALTHY VOLUNTEERS. KCL22 AND K562 CELLS WERE CULTURED AND TREATED WITH DEMETHYLATION DRUGS AND HISTONE DEACETYLASE INHIBITORS. REAL TIME QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC PCR, BISULFITE SEQUENCING PCR, WESTERN BLOTTING, CO-IMMUNOPRECIPITATION AND CHROMATIN IMMUNOPRECIPITATION (CHIP) WAS PERFORMED. PTPN6 WAS DOWN-REGULATED IN CELL LINES AND PATIENTS WITH ADVANCED PHASE CML, WHEREAS DNMT1, DNMT3A, MECP2, MBD2 AND HDAC1 WERE UP-REGULATED. TREATMENT WITH 5-AZACYTIDINE, DECITABINE, SODIUM VALPROATE AND LBH589 INCREASED PTPN6 EXPRESSION, BUT DECREASED THAT OF DNMT1, DNMT3A, MECP2, MBD2 AND HDAC1. IMMUNOPRECIPITATION AND MASS SPECTROMETRY SHOWED THAT HDAC1 COMBINED DIRECTLY WITH PTPN6. CHIP-SEQ SHOWED THAT HDAC1 DID NOT COMBINE WITH THE PROMOTER REGION OF PTPN6, WHILE MAPK, AKT, STAT5, JAK2 AND MYC PROMOTER REGIONS ALL COMBINED WITH HDAC1. PTPN6 IS ASSOCIATED WITH PROGRESSION OF CML. LOW EXPRESSION LEVEL OF PTPN6 WAS ASSOCIATED WITH DNA METHYLATION AND REGULATED BY HISTONE ACETYLATION. HDAC1 PARTICIPATES IN THE REGULATION OF PTPN6. 2017 4 1631 41 DNMT3A METHYLATION IN NEUROPATHIC PAIN. BACKGROUND: MU OPIOID RECEPTOR (MOR) PLAYS A CRUCIAL ROLE IN MEDIATING ANALGESIC EFFECTS OF OPIOIDS AND IS CLOSELY ASSOCIATED WITH THE PATHOLOGIES OF NEUROPATHIC PAIN. PREVIOUS STUDIES HAVE REPORTED THAT PERIPHERAL NERVE INJURY DOWNREGULATES MOR EXPRESSION, BUT THE EPIGENETIC MECHANISMS REMAIN UNKNOWN. OBJECTIVE: THEREFORE, WE INVESTIGATED DNA METHYLTRANSFERASE3A (DNMT3A) EXPRESSION OR METHYLATION CHANGES WITHIN MOR PROMOTER IN THE SPINAL CORD IN A NEUROPATHIC PAIN INDUCED BY A CHRONIC CONSTRICTION INJURY (CCI) MOUSE MODEL AND FURTHER DETERMINED WHETHER THESE INJURY-ASSOCIATED CHANGES ARE REVERSIBLE BY PHARMACOLOGICAL INTERVENTIONS. METHODS: A CCI MOUSE MODEL WAS ESTABLISHED AND TISSUE SPECIMENS OF LUMBAR SPINAL CORDS WERE COLLECTED. THE NOCICEPTION THRESHOLD WAS EVALUATED BY A MODEL HEATED 400 BASE. DNMT3A AND MOR MRNA AND PROTEIN LEVEL WERE DETECTED BY REAL-TIME-POLYMERASE CHAIN REACTION AND WESTERN BLOT, RESPECTIVELY. METHYLATION OF DNMT3A GENE WAS MEASURED BY METHYLATION-SPECIFIC PCR. RESULTS: OUR DATA SHOWED THAT CHRONIC NERVE INJURY LED TO A SIGNIFICANT UPREGULATION OF DNMT3A EXPRESSION THAT WAS ASSOCIATED WITH INCREASED METHYLATION OF MOR GENE PROMOTER AND DECREASED MOR PROTEIN EXPRESSION IN THE SPINAL CORD. INHIBITION OF DNMT3A CATALYTIC ACTIVITY WITH DNMT INHIBITOR RG108 SIGNIFICANTLY BLOCKED THE INCREASE IN METHYLATION OF THE MOR PROMOTER, AND THEN UPREGULATED MOR EXPRESSION AND ATTENUATED THERMAL HYPERALGESIA IN NEUROPATHIC PAIN MICE. CONCLUSION: THIS STUDY DEMONSTRATES THAT AN INCREASE OF DNMT3A EXPRESSION AND MOR METHYLATION EPIGENETICALLY PLAY AN IMPORTANT ROLE IN NEUROPATHIC PAIN. TARGETING DNMT3A TO THE PROMOTER OF MOR GENE BY DNMT INHIBITOR MAY BE A PROMISING APPROACH TO THE DEVELOPMENT OF NEW NEUROPATHIC PAIN THERAPY. 2017 5 6660 37 UPREGULATION OF CXCR4 THROUGH PROMOTER DEMETHYLATION CONTRIBUTES TO INFLAMMATORY HYPERALGESIA IN RATS. AIM AND METHODS: CHRONIC PAIN ASSOCIATED WITH INFLAMMATION IS A COMMON CLINICAL PROBLEM, AND THE UNDERLYING MECHANISMS YET ARE INCOMPLETELY DEFINED. DNA METHYLATION HAS BEEN IMPLICATED IN THE PATHOGENESIS OF CHRONIC PAIN. HOWEVER, THE SPECIFIC GENES REGULATED BY DNA METHYLATION UNDER INFLAMMATORY PAIN CONDITION REMAIN LARGELY UNKNOWN. HERE, WE INVESTIGATED HOW CHEMOKINE RECEPTOR CXCR4 EXPRESSION IS REGULATED BY DNA METHYLATION AND HOW IT CONTRIBUTES TO INFLAMMATORY PAIN INDUCED BY COMPLETE FREUND'S ADJUVANT (CFA) IN RATS. RESULTS: INTRAPLANTAR INJECTION OF CFA COULD NOT ONLY INDUCE SIGNIFICANT HYPERALGESIA IN RATS, BUT ALSO SIGNIFICANTLY INCREASE THE EXPRESSION OF CXCR4 MRNA AND PROTEIN IN THE DORSAL ROOT GANGLION (DRG). INTRATHECAL INJECTION OF CXCR4 ANTAGONIST AMD3100 SIGNIFICANTLY RELIEVED HYPERALGESIA IN INFLAMMATORY RATS IN A TIME- AND DOSE-DEPENDENT MANNER. BISULFITE SEQUENCING AND METHYLATION-SPECIFIC PCR DEMONSTRATE THAT CFA INJECTION LED TO A SIGNIFICANT DEMETHYLATION OF CPG ISLAND AT CXCR4 GENE PROMOTER. CONSISTENTLY, THE EXPRESSION OF DNMT3B WAS SIGNIFICANTLY DOWNREGULATED AFTER CFA INJECTION. ONLINE SOFTWARE PREDICTION REVEALS THREE BINDING SITES OF P65 IN THE CPG ISLAND OF CXCR4 GENE PROMOTER, WHICH HAS CONFIRMED BY THE CHROMATIN IMMUNOPRECIPITATION ASSAY, CFA TREATMENT SIGNIFICANTLY INCREASES THE RECRUITMENT OF P65 TO CXCR4 GENE PROMOTER. INHIBITION OF NF-KB SIGNALING USING P65 INHIBITOR PYRROLIDINE DITHIOCARBAMATE SIGNIFICANTLY PREVENTED THE INCREASES OF THE CXCR4 EXPRESSION. CONCLUSION: UPREGULATION OF CXCR4 EXPRESSION DUE TO PROMOTER DEMETHYLATION FOLLOWED BY INCREASED RECRUITMENT OF P65 TO PROMOTER OF CXCR4 GENE CONTRIBUTES TO INFLAMMATORY HYPERALGESIA. THESE FINDINGS PROVIDE A THEORETICAL BASIS FOR THE TREATMENT OF CHRONIC PAIN FROM AN EPIGENETIC PERSPECTIVE. 2018 6 2300 30 EPIGENETIC REGULATION OF BDNF EXPRESSION IN THE PRIMARY SENSORY NEURONS AFTER PERIPHERAL NERVE INJURY: IMPLICATIONS IN THE DEVELOPMENT OF NEUROPATHIC PAIN. BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) IS KNOWN TO BE UP-REGULATED IN THE DORSAL ROOT GANGLION (DRG) AFTER PERIPHERAL NERVE INJURY, AND TO CONTRIBUTE TO NEUROPATHIC PAIN. HERE, WE FOUND THAT THERMAL HYPERALGESIA AND MECHANICAL ALLODYNIA AT DAY 7 POST-INJURY WERE INHIBITED ONLY WHEN ANTI-BDNF ANTIBODY WAS INTRATHECALLY ADMINISTRATED AT DAY 2 POST-INJURY. CONSISTENT WITH BEHAVIORAL RESULTS, WESTERN BLOT ANALYSIS SHOWED THAT THE EXPRESSION LEVELS OF BDNF PROTEIN IN THE SPINAL DORSAL HORN WERE MARKEDLY INDUCED DURING EARLY STAGE POST-INJURY. MOREOVER, THE MAXIMAL INCREASE IN BDNF MRNA EXPRESSION IN THE DRG WAS OBSERVED AT DAY 1 POST-INJURY, AND SIGNIFICANTLY ELEVATED LEVELS WERE SUSTAINED FOR AT LEAST 14 DAYS. FOUR OF FIVE BDNF MRNA TRANSCRIPTS WERE UP-REGULATED AFTER NERVE INJURY, AND THE MOST INDUCIBLE TRANSCRIPT WAS EXON I. USING A CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY, WE FOUND THAT NERVE INJURY PROMOTES HISTONE H3 AND H4 ACETYLATION, TRANSCRIPTIONALLY ACTIVE MODIFICATIONS, AT BDNF PROMOTER I AT DAY 1 POST-INJURY, AND THE LEVELS OF HISTONE ACETYLATION REMAIN ELEVATED FOR AT LEAST 7 DAYS. TAKEN TOGETHER, OUR FINDINGS SUGGEST THAT AN INITIAL INCREASE IN BDNF EXON I EXPRESSION CONTROLLED BY EPIGENETIC MECHANISMS MIGHT HAVE A CRUCIAL ROLE IN THE DEVELOPMENT OF NEUROPATHIC PAIN. 2013 7 766 36 CCL5 SUPPRESSES KLOTHO EXPRESSION VIA P-STAT3/DNA METHYLTRANSFERASE1-MEDIATED PROMOTER HYPERMETHYLATION. BACKGROUND: ENHANCED INFLAMMATION AND REDUCED KLOTHO ARE COMMON FEATURES IN CHRONIC KIDNEY DISEASE (CKD). INFLAMMATION INDUCES DNA HYPERMETHYLATION. THIS STUDY ASSESSED THE PERFORMANCE OF INFLAMMATORY MARKER C-C MOTIF CHEMOKINE 5 (CCL5) IN EPIGENETIC REGULATION OF KLOTHO EXPRESSION. METHODS: FIFTY CKD PATIENTS AND 25 MATCHED CONTROLS WERE ENROLLED, AND SERUM CCL5 LEVEL, SKLOTHO LEVEL, AND DNA METHYLATION WERE EVALUATED IN THESE SUBJECTS. A RENAL INTERSTITIAL FIBROSIS (RIF) MODEL WITH CKD WAS INDUCED IN MICE VIA UNILATERAL URETERAL OBSTRUCTION (UUO) IN VIVO AND HUMAN PROXIMAL TUBULAR EPITHELIAL (HK-2) CELLS TREATED WITH CCL5 IN VITRO. 5-AZA-2'-DEOXYCYTIDINE (5-AZA), A DNA METHYLTRANSFERASE INHIBITOR WAS GIVEN TO UUO MICE. HEMATOXYLIN AND EOSIN (HE) AND MASSON TRICHROME STAINING WERE ADOPTED TO EVALUATE RENAL PATHOLOGICAL CHANGES. METHYLATION-SPECIFIC PCR WAS PERFORMED TO ASSESS DNA METHYLATION OF KLOTHO PROMOTER IN THE PERIPHERAL BLOOD LEUCOCYTES (PBLS) FROM CKD PATIENTS AND OBSTRUCTIVE KIDNEY FROM UUO MICE. CCL5, KLOTHO, AND DNA METHYLTRANSFERASES (DNMTS) WERE DETERMINED BY ELISAS, IMMUNOFLUORESCENCE, OR WESTERN BLOTTING. HK-2 CELLS WERE EXPOSED TO CCL5 WITH OR WITHOUT 5-AZA AND STATTIC, A P-SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3 (STAT3) INHIBITOR, AND EXPRESSIONS OF P-STAT3, DNMT1, AND KLOTHO WERE DETERMINED BY WESTERN BLOTTING. RESULTS: CCL5 UPREGULATION CONCOMITANT WITH KLOTHO DOWNREGULATION IN SERUM AND GLOBAL DNA METHYLATION IN PBLS WERE OBSERVED IN CKD SAMPLES. UUO CONTRIBUTED TO SEVERE RENAL INTERSTITIAL FIBROSIS AND ENHANCED EXPRESSIONS OF FIBROTIC MARKERS. MOREOVER, UUO INCREASED THE CCL5 LEVEL, INDUCED KLOTHO PROMOTER METHYLATION, SUPPRESSED KLOTHO LEVEL, ACTIVATED P-STAT3 SIGNALING, AND UPREGULATED DNMT1 LEVEL. A SIMILAR OBSERVATION WAS MADE IN HK-2 CELLS TREATED WITH CCL5. MORE IMPORTANTLY, 5-AZA INHIBITED UUO-INDUCED KLOTHO HYPERMETHYLATION, REVERSED KLOTHO, DOWNREGULATED P-STAT3 EXPRESSIONS, AND AMELIORATED RIF IN VIVO. THE CONSISTENT FINDINGS IN VITRO WERE ALSO OBTAINED IN HK-2 CELLS EXPOSED TO 5-AZA AND STATTIC. CONCLUSION: THE CCL5/P-STAT3/DNMT1 AXIS IS IMPLICATED IN EPIGENETIC REGULATION OF KLOTHO EXPRESSION IN CKD. THIS STUDY PROVIDES NOVEL THERAPEUTIC POSSIBILITIES FOR REVERSAL OF KLOTHO SUPPRESSION BY CKD. 2022 8 1320 38 DEMETHYLATION REGULATION OF BDNF GENE EXPRESSION IN DORSAL ROOT GANGLION NEURONS IS IMPLICATED IN OPIOID-INDUCED PAIN HYPERSENSITIVITY IN RATS. REPEATED ADMINISTRATION OF MORPHINE MAY RESULT IN OPIOID-INDUCED HYPERSENSITIVITY (OIH), WHICH INVOLVES ALTERED EXPRESSION OF NUMEROUS GENES, INCLUDING BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) IN DORSAL ROOT GANGLION (DRG) NEURONS. YET, IT REMAINS UNCLEAR HOW BDNF EXPRESSION IS INCREASED IN DRG NEURONS AFTER REPEATED MORPHINE TREATMENT. DNA METHYLATION IS AN IMPORTANT MECHANISM OF EPIGENETIC CONTROL OF GENE EXPRESSION. IN THE CURRENT STUDY, WE HYPOTHESIZED THAT THE DEMETHYLATION REGULATION OF CERTAIN BDNF GENE PROMOTERS IN DRG NEURONS MAY CONTRIBUTE TO THE DEVELOPMENT OF OIH. REAL-TIME RT-PCR WAS USED TO ASSESS CHANGES IN THE MRNA TRANSCRIPTION LEVELS OF MAJOR BDNF EXONS INCLUDING EXON I, II, IV, VI, AS WELL AS TOTAL BDNF MRNA IN DRGS FROM RATS AFTER REPEATED MORPHINE ADMINISTRATION. THE LEVELS OF EXON IV AND TOTAL BDNF MRNA WERE SIGNIFICANTLY UPREGULATED BY REPEATED MORPHINE ADMINISTRATION, AS COMPARED TO THAT IN SALINE CONTROL GROUP. FURTHER, ELISA ARRAY AND IMMUNOCYTOCHEMISTRY STUDY REVEALED A ROBUST UPREGULATION OF BDNF PROTEIN EXPRESSION IN DRG NEURONS AFTER REPEATED MORPHINE EXPOSURE. CORRESPONDINGLY, THE METHYLATION LEVELS OF BDNF EXON IV PROMOTER SHOWED A SIGNIFICANT DOWNREGULATION BY MORPHINE TREATMENT. IMPORTANTLY, INTRATHECAL ADMINISTRATION OF A BDNF ANTIBODY, BUT NOT CONTROL IGG, SIGNIFICANTLY INHIBITED MECHANICAL HYPERSENSITIVITY THAT DEVELOPED IN RATS AFTER REPEATED MORPHINE TREATMENT. CONVERSELY, INTRATHECAL ADMINISTRATION OF AN INHIBITOR OF DNA METHYLATION, 5-AZA-2'-DEOXYCYTIDINE (5-AZA-DC) MARKEDLY UPREGULATED THE BDNF PROTEIN EXPRESSION IN DRG NEURONS AND ENHANCED THE MECHANICAL ALLODYNIA AFTER REPEATED MORPHINE EXPOSURE. TOGETHER, OUR FINDINGS SUGGEST THAT DEMETHYLATION REGULATION OF BDNF GENE PROMOTER MAY BE IMPLICATED IN THE DEVELOPMENT OF OIH THROUGH EPIGENETIC CONTROL OF BDNF EXPRESSION IN DRG NEURONS. 2016 9 3082 40 GENOME-WIDE REDISTRIBUTION OF MECP2 IN DORSAL ROOT GANGLIA AFTER PERIPHERAL NERVE INJURY. BACKGROUND: METHYL-CPG-BINDING PROTEIN 2 (MECP2), A PROTEIN WITH AFFINITY FOR METHYLATED CYTOSINES, IS CRUCIAL FOR NEURONAL DEVELOPMENT AND FUNCTION. MECP2 REGULATES GENE EXPRESSION THROUGH ACTIVATION, REPRESSION AND CHROMATIN REMODELING. MUTATIONS IN MECP2 CAUSE RETT SYNDROME, AND THESE PATIENTS DISPLAY IMPAIRED NOCICEPTION. WE OBSERVED AN INCREASE IN MECP2 EXPRESSION IN MOUSE DORSAL ROOT GANGLIA (DRG) AFTER PERIPHERAL NERVE INJURY. THE FUNCTIONAL IMPLICATION OF INCREASED MECP2 IS LARGELY UNKNOWN. TO IDENTIFY REGIONS OF THE GENOME BOUND BY MECP2 IN THE DRG AND THE CHANGES INDUCED BY NERVE INJURY, A CHROMATIN IMMUNOPRECIPITATION OF MECP2 FOLLOWED BY SEQUENCING (CHIP-SEQ) WAS PERFORMED 4 WEEKS AFTER SPARED NERVE INJURY (SNI). RESULTS: WHILE THE NUMBER OF BINDING SITES ACROSS THE GENOME REMAINED SIMILAR IN THE SNI MODEL AND SHAM CONTROL, SNI INDUCED THE REDISTRIBUTION OF MECP2 TO TRANSCRIPTIONALLY RELEVANT REGIONS. TO DETERMINE HOW DIFFERENTIAL BINDING OF MECP2 CAN AFFECT GENE EXPRESSION IN THE DRG, WE INVESTIGATED MMU-MIR-126, A MICRORNA LOCUS THAT HAD ENRICHED MECP2 BINDING IN THE SNI MODEL. ENRICHED MECP2 BINDING TO MIR-126 LOCUS AFTER NERVE INJURY REPRESSED MIR-126 EXPRESSION, AND THIS WAS NOT MEDIATED BY ALTERATIONS IN METHYLATION PATTERN AT THE MIR-126 LOCUS. DOWNREGULATION OF MIR-126 RESULTED IN THE UPREGULATION OF ITS TWO TARGET GENES DNMT1 AND VEGFA IN NEURO 2A CELLS AND IN SNI MODEL COMPARED TO CONTROL. THESE TARGET GENES WERE SIGNIFICANTLY DOWNREGULATED IN MECP2-NULL MICE COMPARED TO WILD-TYPE LITTERMATES, INDICATING A REGULATORY ROLE FOR MECP2 IN ACTIVATING DNMT1 AND VEGFA EXPRESSION. INTRATHECAL DELIVERY OF MIR-126 WAS NOT SUFFICIENT TO REVERSE NERVE INJURY-INDUCED MECHANICAL AND THERMAL HYPERSENSITIVITY, BUT DECREASED DNMT1 AND VEGFA EXPRESSION IN THE DRG. CONCLUSIONS: OUR STUDY SHOWS A REGULATORY ROLE FOR MECP2 IN THAT CHANGES IN GLOBAL REDISTRIBUTION CAN RESULT IN DIRECT AND INDIRECT MODULATION OF GENE EXPRESSION IN THE DRG. ALTERATIONS IN GENOME-WIDE BINDING OF MECP2 THEREFORE PROVIDE A MOLECULAR BASIS FOR A BETTER UNDERSTANDING OF EPIGENETIC REGULATION-INDUCED MOLECULAR CHANGES UNDERLYING NERVE INJURY. 2016 10 3459 31 HYPOMETHYLATION OF NERVE GROWTH FACTOR (NGF) PROMOTES BINDING OF C/EBPALPHA AND CONTRIBUTES TO INFLAMMATORY HYPERALGESIA IN RATS. BACKGROUND: CHRONIC PAIN USUALLY ACCOMPANIED BY TISSUE DAMAGE AND INFLAMMATION. HOWEVER, THE PATHOGENESIS OF CHRONIC PAIN REMAINS UNCLEAR. METHODS: WE INVESTIGATED THE ROLE OF NERVE GROWTH FACTOR (NGF) IN CHRONIC INFLAMMATORY PAIN INDUCED BY COMPLETE FREUND'S ADJUVANT (CFA), EXPLORED THE METHYLATION STATUS OF CPG ISLANDS IN THE PROMOTER REGION OF THE NGF GENE, AND CLARIFIED THE FUNCTION AND MECHANISM OF C/EBPALPHA-NGF SIGNALING PATHWAY FROM EPIGENETIC PERSPECTIVE IN THE CHRONIC INFLAMMATORY PAIN MODEL. RESULTS: CFA INDUCED SIGNIFICANT HYPERALGESIA AND CONTINUOUS UPREGULATION OF NGF MRNA AND PROTEIN LEVELS IN THE L4-6 DORSAL ROOT GANGLIONS (DRGS) IN RATS. HYPOMETHYLATION OF CPG ISLANDS OCCURRED IN THE NGF GENE PROMOTER REGION AFTER CFA TREATMENT. AT THE SAME TIME, THE MIR-29B EXPRESSION LEVEL WAS SIGNIFICANTLY INCREASED, WHILE THE DNA METHYLTRANSFERASE 3B (DNMT3B) LEVEL REDUCED SIGNIFICANTLY. MOREOVER, CFA TREATMENT PROMOTED BINDING OF C/EBPALPHA TO THE NGF GENE PROMOTER REGION AND C/EBPALPHA SIRNA TREATMENT OBVIOUSLY DECREASED EXPRESSION OF NGF LEVELS AND ALSO ALLEVIATE INFLAMMATORY HYPERALGESIA SIGNIFICANTLY IN RATS. CONCLUSION: COLLECTIVELY, THE RESULTS INDICATED THAT CFA LEADS TO THE UPREGULATION OF MIR-29B LEVEL, WHICH REPRESSES THE EXPRESSION OF DNMT3B, ENHANCES THE DEMETHYLATION OF THE NGF GENE PROMOTER REGION, AND PROMOTES THE BINDING OF C/EBPALPHA WITH THE NGF GENE PROMOTER, THUS RESULTS IN THE UPREGULATION OF NGF GENE EXPRESSION AND MAINTENANCE OF CHRONIC INFLAMMATORY PAIN. 2020 11 3832 28 INVOLVEMENT OF SPINAL SIRT1 IN DEVELOPMENT OF CHRONIC CONSTRICTION INJURY INDUCED NEUROPATHIC PAIN IN RATS. IT IS KNOWN THAT THE EPIGENETIC PROCESS OF HISTONE ACETYLATION IS INVOLVED IN THE NEUROPATHIC PAIN. THE AIM OF THIS STUDY WAS TO DETERMINE WHETHER SIRTUIN TYPE 1 (SIRT1), AN NAD(+) DEPENDENT DEACETYLASE, AFFECTED ALLODYNIA AND HYPERALGESIA IN NEUROPATHIC PAIN. THE NEUROPATHIC PAIN MODEL WAS ESTABLISHED BY LIGATURE OF THE RIGHT SCIATIC NERVE TO INDUCE CHRONIC CONSTRICTION INJURY (CCI) IN RATS. HISTONE ACETYLTRANSFERASE (HAT) ACTIVITY WAS INCREASED AND, AND HISTONE DEACETYLASE (HDAC) ACTIVITY WAS DECLINED IN TISSUE OF THE SPINAL DORSA HORN IN CCI RATES BY MEANS OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA). THE PERSISTENT HYPERALGESIA AND ALLODYNIA CAUSED BY CCI WERE ASSOCIATED WITH DOWNREGULATION OF SIRT1 AND UPREGULATION OF ACETYLATED-H3 (AC-H3) IN TISSUE OF THE SPINAL CORD BY WESTERN BLOT ASSAY, WHICH WAS REVERSED AFTER INTRATHECAL INJECTION OF SIRT1 AGONIST SRT1720. SRT1720 TREATMENT ACHIEVED ANALGESIC THROUGH INHIBITING THE ACETYLATION OF NUCLEAR FACTOR KAPPA B (NF-KAPPAB) AND BLOCKING THE RELEASES OF THE INFLAMMATORY FACTORS INCLUDING TUMOR NECROSIS FACTOR-ALPHA (TNF-ALPHA) AND INTERLEUKIN (IL)-6 BY MEANS OF WESTERN BLOT AND REAL-TIME QUANTITATIVE PCR (RT-PCR), RESPECTIVELY. TAKEN TOGETHER, THESE DATA SUGGEST THAT SIRT1 IN THE SPINAL CORD PLAYS AN IMPORTANT ROLE IN THE NEUROPATHIC PAIN IN THE RAT MODEL. 2018 12 5479 40 RESVERATROL ATTENUATES CIGARETTE SMOKE EXTRACT INDUCED CELLULAR SENESCENCE IN HUMAN AIRWAY EPITHELIAL CELLS BY REGULATING THE MIR-34A/SIRT1/NF-KAPPAB PATHWAY. CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) IS CHARACTERIZED BY ACCELERATED LUNG AGING. SMOKING IS THE CRITICAL RISK FACTOR FOR COPD. CELLULAR SENESCENCE OF AIRWAY EPITHELIAL CELLS IS THE CYTOLOGICAL BASIS OF ACCELERATED LUNG AGING IN COPD, AND THE REGULATION OF MICRORNAS (MIRNAS) IS THE CENTRAL EPIGENETIC MECHANISM OF CELLULAR SENESCENCE. RESVERATROL (RES) IS A POLYPHENOL WITH ANTI-AGING PROPERTIES. THIS STUDY INVESTIGATED WHETHER RES ATTENUATES CIGARETTE SMOKE EXTRACT (CSE)-INDUCED CELLULAR SENESCENCE IN HUMAN AIRWAY EPITHELIAL CELLS (BEAS-2B) THROUGH THE MIR-34A/SIRT1/NUCLEAR FACTOR-KAPPAB (NF-KAPPAB) PATHWAY. BEAS-2B CELLS WERE TREATED WITH RES, CSE AND TRANSFECTED WITH MIR-34A-5P MIMICS. CELLULAR SENESCENCE WAS EVALUATED BY SENESCENCE -RELATED BETA-GALACTOSIDASE (SA-BETA-GAL) STAINING AND EXPRESSION OF SENESCENCE-RELATED GENES (P16, P21, AND P53). THE EXPRESSIONS OF MIR-34A-5P, SIRT1, AND NF-KAPPAB P65 WERE EXAMINED USING QUANTITATIVE REAL TIME POLYMERASE CHAIN REACTION AND WESTERN BLOTTING. THE SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE (SASP) CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) WERE ASSESSED BY ENZYME-LINKED IMMUNOSORBENT ASSAY. THE BINDING BETWEEN MIR-34A-5P AND SIRT1 WAS CONFIRMED BY DUAL-LUCIFERASE REPORTER ASSAY. THE RESULTS SHOWED THAT CSE DOSE-DEPENDENTLY DECREASED CELL VIABILITY AND ELEVATED CELLULAR SENESCENCE, CHARACTERIZED BY INCREASED SA-BETA-GAL STAINING AND SENESCENCE-RELATED GENE EXPRESSIONS (P16, P21, AND P53). FURTHER, CSE DOSE-DEPENDENTLY INCREASED THE EXPRESSION OF MIR-34A-5P AND SASP CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) IN BEAS-2B CELLS. PRETREATMENT WITH RES INHIBITED CSE-INDUCED CELLULAR SENESCENCE AND SECRETION OF SASP CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) IN A DOSE-DEPENDENT MANNER. MOREOVER, RES REVERSED THE CSE-INDUCED DOWN-REGULATION OF SIRT1 AND UP-REGULATION OF MIR-34A-5P AND NF-KAPPAB P65. SIRT1 IS A TARGET OF MIR-34A-5P. OVEREXPRESSION OF MIR-34A-5P VIA TRANSFECTION WITH MIR-34A-5P MIMIC IN BEAS-2B CELLS ATTENUATED THE INHIBITORY EFFECT OF RES ON CELLULAR SENESCENCE, ACCOMPANIED BY REVERSING THE EXPRESSION OF SIRT1 AND NF-KAPPAB P65. IN CONCLUSION, RES ATTENUATED CSE-INDUCED CELLULAR SENESCENCE IN BEAS-2B CELLS BY REGULATING THE MIR-34A/SIRT1/NF-KAPPAB PATHWAY, WHICH MAY PROVIDE A NEW APPROACH FOR COPD TREATMENT. 2022 13 2197 24 EPIGENETIC MODIFICATION OF BDNF MEDIATES NEUROPATHIC PAIN VIA MIR-30A-3P/EP300 AXIS IN CCI RATS. RECENT INVESTIGATION OF MICRORNAS ON CHRONIC PAIN HAS DEVELOPED A BREAKTHROUGH IN NEUROPATHIC PAIN MANAGEMENT. IN THE PRESENT STUDY, DECREASED EXPRESSION OF MIR-30A-3P WAS REPORTED USING QRT-PCR ANALYSIS AND LOSS OF MIR-30A-3P PROMOTED NEUROPATHIC PAIN PROGRESSION IN SCIATIC NERVE CHRONIC CONSTRICTIVE INJURY RATS THROUGH DETERMINING THE PAIN THRESHOLD. WE PREDICTED MIR-30A-3P COULD TARGET E-CADHERIN TRANSCRIPTIONAL ACTIVATOR (EP300) VIA BIOINFORMATICS ANALYSIS. MEANWHILE, WE FOUND THAT BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) IS INVOLVED IN NEUROPATHIC PAIN. HERE, WE EXHIBITED THAT EP300 EPIGENETICALLY UP-REGULATED BDNF VIA ENHANCING ACETYLATED HISTONE H3 AND H4 ON THE PROMOTER. FOR ANOTHER, MIR-30A-3P WAS ABLE TO MODIFY THE LEVEL OF BDNF AND ACETYLATED HISTONE H3 AND H4. LOSS OF MIR-30A-3P ENHANCED EP300 AND BDNF COLOCALIZATION IN CCI RATS. SUBSEQUENTLY, IT WAS SHOWN THAT INCREASED EP300 INDUCED NEUROPATHIC PAIN BY AN ENHANCEMENT OF NEURONAL BDNF LEVEL IN VIVO. TO SUM UP, IT WAS REVEALED THAT EPIGENETIC MODIFICATION OF BDNF PROMOTED NEUROPATHIC PAIN VIA EP300 INDUCED BY MIR-30A-3P IN CCI RATS. 2020 14 1620 34 DNA METHYLTRANSFERASE-MEDIATED TRANSCRIPTIONAL SILENCING IN MALIGNANT GLIOMA: A COMBINED WHOLE-GENOME MICROARRAY AND PROMOTER ARRAY ANALYSIS. EPIGENETIC INACTIVATION OF TUMOR SUPPRESSOR GENES IS A COMMON FEATURE IN HUMAN CANCER. PROMOTER HYPERMETHYLATION AND HISTONE DEACETYLATION ARE REVERSIBLE EPIGENETIC MECHANISMS ASSOCIATED WITH TRANSCRIPTIONAL REGULATION. DNA METHYLTRANSFERASES (DNMT1 AND DNMT3B) REGULATE AND MAINTAIN PROMOTER METHYLATION AND ARE OVEREXPRESSED IN HUMAN CANCER. WE PERFORMED WHOLE-GENOME MICROARRAY ANALYSIS TO IDENTIFY GENES WITH ALTERED EXPRESSION AFTER RNAI-INDUCED SUPPRESSION OF DNMT IN A GLIOBLASTOMA MULTIFORME (GBM) CELL LINE. WE THEN IDENTIFIED GENES WITH BOTH DECREASED EXPRESSION AND EVIDENCE OF PROMOTER CPG ISLAND HYPERMETHYLATION IN GBM TISSUE SAMPLES USING A COMBINED WHOLE-GENOME MICROARRAY TRANSCRIPTOME ANALYSIS IN CONJUNCTION WITH A PROMOTER ARRAY ANALYSIS AFTER DNA IMMUNOPRECIPITATION WITH ANTI-5-METHYLCYTIDINE. DNMT1 AND 3B KNOCKDOWN RESULTED IN THE RESTORED EXPRESSION OF 308 GENES THAT ALSO CONTAINED PROMOTER REGION HYPERMETHYLATION. OF THESE, 43 WERE ALSO FOUND TO BE DOWNREGULATED IN GBM TISSUE SAMPLES. THREE DOWNREGULATED GENES WITH HYPERMETHYLATED PROMOTERS AND RESTORED EXPRESSION IN RESPONSE TO ACUTE DNMT SUPPRESSION WERE ASSAYED FOR METHYLATION CHANGES USING BISULFITE SEQUENCE ANALYSIS OF THE PROMOTER REGION AFTER CHRONIC DNMT SUPPRESSION. RESTORATION OF GENE EXPRESSION WAS NOT ASSOCIATED WITH CHANGES IN PROMOTER REGION METHYLATION, BUT RATHER WITH CHANGES IN HISTONE METHYLATION AND CHROMATIN CONFORMATION. TWO OF THE IDENTIFIED GENES EXHIBITED GROWTH SUPPRESSIVE ACTIVITY IN IN VITRO ASSAYS. COMBINING TARGETED GENETIC MANIPULATIONS WITH COMPREHENSIVE GENOMIC AND EXPRESSION ANALYSES PROVIDES A POTENTIALLY POWERFUL NEW APPROACH FOR IDENTIFYING EPIGENETICALLY REGULATED GENES IN GBM. 2009 15 6231 54 THE LONG NONCODING RNA MEG3 AND ITS TARGET MIR-147 REGULATE JAK/STAT PATHWAY IN ADVANCED CHRONIC MYELOID LEUKEMIA. BACKGROUND: LONG NON-CODING (LNC) RNAS PLAYS AN IMPORTANT ROLE IN CHRONIC MYELOID LEUKEMIA (CML). IN THIS STUDY, WE AIMED TO UNCOVER THE MECHANISM OF THE LNCRNA MATERNALLY EXPRESSED 3 (MEG3) AND ITS TARGET MICRORNA-147 (MIR-147) IN CML. METHODS: SIXTY CML PATIENTS AND 10 HEALTHY DONORS WERE INCLUDED IN THE STUDY. THE METHYLATION OF MEG3 AND MIR-147 PROMOTER WAS DETERMINED BY METHYLATION-SPECIFIC PCR. THE RELATIONSHIP OF MEG3 AND MIR-147 WAS EXPLORED BY LUCIFERASE ASSAY. THE INTERACTIONS OF PROTEINS WERE STUDIED BY RNA PULL-DOWN ASSAY, RNA IMMUNOPRECIPITATION AND CO-IMMUNOPRECIPITATION. FINDINGS: PATIENTS IN ACCELERATED PHASE CML (CML-AP) AND BLAST PHASE CML (CML-BP) SHOWED LOWER EXPRESSIONS OF MEG3 AND MIR-147 AND HIGHER EXPRESSIONS OF DNMT1, DNMT3B, MBD2, MECP2 AND HDAC1 COMPARED TO THE CONTROLS. THESE PATIENTS ALSO SHOWED A HIGHER DEGREE OF METHYLATION OF MEG3 AND MIR-147 WHILE THERE WAS A REDUCTION AFTER CHIDAMIDE TREATMENT. FURTHERMORE, THE OVEREXPRESSION OF MEG3 AND MIR-147 INHIBITED CELL PROLIFERATION BOTH IN VIVO AND IN VITRO, PROMOTED APOPTOSIS AND DECREASED THE EXPRESSIONS OF DNMT1, DNMT3A, DNMT3B, MBD2, HDAC1 AND MECP2. WE ALSO FOUND MEG3 INTERACTED WITH DNMT1, JAK2, STAT3, HDAC1, AND TYK2, AND JAK2 WAS BOUND TO STAT3, STAT5 AND MYC. MORE INTERESTINGLY, JAK2 WAS BOUND TO TYK2 BY THE BRIDGE OF MEG3. INTERPRETATION: LNCRNA MEG3 AND ITS TARGET MIR-147 MAY SERVE AS A NOVEL THERAPEUTIC TARGET FOR CML BLAST CRISIS, AND CHIDAMIDE MIGHT HAVE A POTENTIAL CLINICAL APPLICATION IN TREATING CML BLAST CRISIS. 2018 16 4579 21 N(6)-METHYLADENOSINE METHYLASE METTL3 CONTRIBUTES TO NEUROPATHIC PAIN BY EPIGENETIC SILENCING OF MU OPIOID RECEPTOR. WE AIMED AT EXPLORING THE ROLE AND MECHANISM OF METTL3-MEDIATED M(6)A MODIFICATION IN NEUROPATHIC PAIN. MALE SPRAGUE-DAWLEY RATS WERE RANDOMLY DIVIDED INTO FOUR GROUPS: SHAM OPERATION GROUP (SHAM GROUP), CHRONIC CONSTRICTION INJURY (CCI) OF THE SCIATIC NERVE MODEL GROUP (NPP GROUP), INTRATHECAL INJECTION OF VIRUS DOWN-REGULATED METTL3 + CCI MODEL GROUP (M3 + NPP GROUP) AND INTRATHECAL INJECTION OF NEGATIVE CONTROL VIRUS + CCI MODEL GROUP (SCR + NPP GROUP). THE M3 + NPP GROUP AND THE SCR + NPP GROUP WERE INTRATHECALLY INJECTED WITH VIRUS NINETEEN DAYS BEFORE OPERATION. THE PAW WITHDRAWAL MECHANICAL THRESHOLDS AND PAW WITHDRAWAL LATENCY WERE RESPECTIVELY RECORDED ONE DAY BEFORE OPERATION, THREE DAYS, FIVE DAYS AND SEVEN DAYS AFTER OPERATION. THE RATS WERE SACRIFICED ON THE SEVENTH DAY AFTER OPERATION, AND THEIR SPINAL CORD TISSUES WERE TAKEN. THE FROZEN SECTIONS OF RATS WERE PERFORMED TO OBSERVE THE EXPRESSION OF GREEN FLUORESCENT PROTEIN OF THE VIRUS. THE METHYLATION LEVEL OF RNA, THE PROTEIN EXPRESSION OF M(6)A-RELATED ENZYME (METTL3) AND MU OPIOID RECEPTOR (MOR) IN SPINAL CORD TISSUES OF THE FOUR GROUPS WERE MEASURED. DOWNREGULATION OF METTL3 HAD NO EFFECT ON THE OVERALL METHYLATION LEVEL OF THE SPINAL CORD, BUT IT COULD REGULATE THE METHYLATION LEVEL OF THE OPRM1 GENE RNA ENCODING MOR, PARTIALLY RESTORE THE EXPRESSION OF MOR, AND RELIEVE PAIN IN RATS. IN THE PROCESS OF NPP, METTL3 MAY INHIBIT THE EXPRESSION OF MOR BY REGULATING THE METHYLATION LEVEL OF OPRM1 GENE RNA ENCODING MOR, AND ULTIMATELY PROMOTE THE OCCURRENCE AND DEVELOPMENT OF NPP. 2023 17 3175 36 H2AX PHOSPHORYLATION REGULATED BY P38 IS INVOLVED IN BIM EXPRESSION AND APOPTOSIS IN CHRONIC MYELOGENOUS LEUKEMIA CELLS INDUCED BY IMATINIB. INCREASING EVIDENCE SUGGESTS THAT HISTONE H2AX PLAYS A CRITICAL ROLE IN REGULATION OF TUMOR CELL APOPTOSIS AND ACTS AS A NOVEL HUMAN TUMOR SUPPRESSOR PROTEIN. HOWEVER, THE ACTION OF H2AX IN CHRONIC MYELOGENOUS LEUKEMIA (CML) CELLS IS UNKNOWN. THE DETAILED MECHANISM AND EPIGENETIC REGULATION BY H2AX REMAIN ELUSIVE IN CANCER CELLS. HERE, WE REPORT THAT H2AX WAS INVOLVED IN APOPTOSIS OF CML CELLS. OVEREXPRESSION OF H2AX INCREASED APOPTOTIC SENSITIVITY OF CML CELLS (K562) INDUCED BY IMATINIB. HOWEVER, OVEREXPRESSION OF SER139-MUTATED H2AX (BLOCKING PHOSPHORYLATION) DECREASED SENSITIVITY OF K562 CELLS TO APOPTOSIS. SIMILARLY, KNOCKDOWN OF H2AX MADE K562 CELLS RESISTANT TO APOPTOTIC INDUCTION. THESE RESULTS REVEALED THAT THE FUNCTION OF H2AX INVOLVED IN APOPTOSIS IS STRICTLY RELATED TO ITS PHOSPHORYLATION (SER139). OUR DATA FURTHER INDICATED THAT IMATINIB MAY STIMULATE MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) FAMILY MEMBER P38, AND H2AX PHOSPHORYLATION FOLLOWED A SIMILAR TIME COURSE, SUGGESTING A PARALLEL RESPONSE. H2AX PHOSPHORYLATION CAN BE BLOCKED BY P38 SIRNA OR ITS INHIBITOR. THESE DATA DEMONSTRATED THAT H2AX PHOSPHORYLATION WAS REGULATED BY P38 MAPK PATHWAY IN K562 CELLS. HOWEVER, THE P38 MAPK DOWNSTREAM, MITOGEN- AND STRESS-ACTIVATED PROTEIN KINASE-1 AND -2, WHICH PHOSPHORYLATED HISTONE H3, WERE NOT REQUIRED FOR H2AX PHOSPHORYLATION DURING APOPTOSIS. FINALLY, WE PROVIDED EPIGENETIC EVIDENCE THAT H2AX PHOSPHORYLATION REGULATED APOPTOSIS-RELATED GENE BIM EXPRESSION. BLOCKING OF H2AX PHOSPHORYLATION INHIBITED BIM GENE EXPRESSION. TAKEN TOGETHER, THESE DATA DEMONSTRATED THAT H2AX PHOSPHORYLATION REGULATED BY P38 IS INVOLVED IN BIM EXPRESSION AND APOPTOSIS IN CML CELLS INDUCED BY IMATINIB. 2014 18 3531 39 IMATINIB CAUSES EPIGENETIC ALTERATIONS OF PTEN GENE VIA UPREGULATION OF DNA METHYLTRANSFERASES AND POLYCOMB GROUP PROTEINS. WE HAVE RECENTLY REPORTED THE POSSIBLE IMATINIB-RESISTANT MECHANISM; LONG-TERM EXPOSURE OF LEUKEMIA CELLS TO IMATINIB DOWNREGULATED LEVELS OF PHOSPHATASE AND TENSIN HOMOLOG DELETED ON CHROMOSOME 10 (PTEN) VIA HYPERMETHYLATION OF ITS PROMOTER REGION (LEUKEMIA 2010; 24: 1631). THE PRESENT STUDY EXPLORED THE MOLECULAR MECHANISMS BY WHICH IMATINIB CAUSED METHYLATION ON THE PROMOTER REGION OF THIS TUMOR SUPPRESSOR GENE IN LEUKEMIA CELLS. REAL-TIME REVERSE TRANSCRIPTION PCR FOUND THAT LONG-TERM EXPOSURE OF CHRONIC EOSINOPHILIC LEUKEMIA EOL-1 CELLS EXPRESSING FIP1L1/PLATELET-DERIVED GROWTH FACTOR RECEPTOR-ALPHA TO IMATINIB INDUCED EXPRESSION OF DNA METHYLTRANSFERASE 3A (DNMT3A) AND HISTONE-METHYLTRANSFERASE ENHANCER OF ZESTE HOMOLOG 2 (EZH2), A FAMILY OF POLYCOMB GROUP, THEREBY INCREASING METHYLATION OF THE GENE. IMMUNOPRECIPITATION ASSAY FOUND THE INCREASED COMPLEX FORMATION OF DNMT3A AND EZH2 PROTEINS IN THESE CELLS. MOREOVER, CHROMATIN IMMUNOPRECIPITATION ASSAY SHOWED THAT AMOUNTS OF BOTH DNMT3A AND EZH2 PROTEINS BOUND AROUND THE PROMOTER REGION OF PTEN GENE WERE INCREASED IN EOL-1 CELLS AFTER EXPOSURE TO IMATINIB. FURTHERMORE, WE FOUND THAT LEVELS OF DNMT3A AND EZH2 WERE STRIKINGLY INCREASED IN LEUKEMIA CELLS ISOLATED FROM INDIVIDUALS WITH CHRONIC MYELOGENOUS LEUKEMIA (N=1) AND PHILADELPHIA CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (N=2), WHO RELAPSED AFTER TREATMENT WITH IMATINIB COMPARED WITH THOSE ISOLATED AT THEIR INITIAL PRESENTATION. TAKEN TOGETHER, IMATINIB COULD CAUSE DRUG-RESISTANCE VIA RECRUITMENT OF POLYCOMB GENE COMPLEX TO THE PROMOTER REGION OF THE PTEN AND DOWNREGULATION OF THIS GENE'S TRANSCRIPTS IN LEUKEMIA PATIENTS. 2011 19 6238 45 THE MALIGNANCY SUPPRESSION ROLE OF MIR-23A BY TARGETING THE BCR/ABL ONCOGENE IN CHROMIC MYELOID LEUKEMIA. THE AIM OF THIS STUDY WAS TO INVESTIGATE THE ROLE AND MECHANISM OF MIR-23A IN THE REGULATION OF BCR/ABL AND TO PROVIDE A NEW PROGNOSTIC BIOMARKER FOR CHRONIC MYELOID LEUKEMIA (CML). THE EXPRESSION LEVELS OF MIR-23A AND BCR/ABL WERE ASSESSED IN 42 NEWLY DIAGNOSED CML PATIENTS, 37 CML PATIENTS IN FIRST COMPLETE REMISSION AND 25 HEALTHY CONTROLS. QUANTITATIVE REAL-TIME PCR, WESTERN BLOT ANALYSIS AND COLONY FORMATION ASSAY WERE USED TO EVALUATE CHANGES INDUCED BY OVEREXPRESSION OR INHIBITION OF MIR-23A OR BCR/ABL. MIR-23A MIMIC OR NEGATIVE CONTROL MIMIC WAS TRANSFECTED INTO A CML CELL LINE (K562) AND TWO LUNG CANCER CELL LINES (H157 AND SKMES1) USING LIPOFECTAMINE 2000, AND THE CELLS WERE USED FOR REAL-TIME REVERSE TRANSCRIPTION-PCR (RT-PCR) AND WESTERN BLOT ANALYSIS. WE FOUND THAT THE DOWNREGULATION OF MIR-23A EXPRESSION WAS A FREQUENT EVENT IN BOTH LEUKEMIA CELL LINES AND PRIMARY LEUKEMIC CELLS FROM PATIENTS WITH DE NOVO CML. THE MICROARRAY RESULTS SHOWED THAT MOST OF THE CML PATIENTS EXPRESSED HIGH LEVELS OF BCR/ABL AND LOW LEVELS OF MIR-23A. REAL-TIME RT-PCR AND WESTERN BLOT ANALYSIS SHOWED THAT THE BCR/ABL LEVELS IN MIR-23A-TRANSFECTED CELLS WERE LOWER THAN THOSE IN THE CONTROL GROUPS. ECTOPIC EXPRESSION OF MIR-23A IN K562 CELLS LED TO CELLULAR SENESCENCE. MOREOVER, WHEN K562 CELLS WERE TREATED WITH 5-AZA-2'-DEOXYCYTIDINE, A DNA METHYLATION INHIBITOR, BCR/ABL EXPRESSION WAS UPREGULATED, WHICH INDICATES EPIGENETIC SILENCING OF MIR-23A IN LEUKEMIC CELLS. BCR/ABL AND MIR-23A EXPRESSIONS WERE INVERSELY RELATED TO CML, AND BCR/ABL EXPRESSION WAS REGULATED BY MIR-23A IN LEUKEMIC CELLS. THE EPIGENETIC SILENCING OF MIR-23A LED TO DEREPRESSION OF BCR/ABL EXPRESSION, AND CONSEQUENTLY CONTRIBUTES TO CML DEVELOPMENT AND PROGRESSION. 2014 20 6235 37 THE M(6)A DEMETHYLASE FTO PROMOTES RENAL EPITHELIAL-MESENCHYMAL TRANSITION BY REDUCING THE M(6)A MODIFICATION OF LNCRNA GAS5. BACKGROUND: RENAL INTERSTITIAL FIBROSIS (RIF) IS THE MAIN PATHOLOGICAL CHANGE OF A VARIETY OF CHRONIC KIDNEY DISEASES (CKD). EPIGENETIC MODIFICATIONS OF FIBROSIS-PRONE GENES REGULATE RIF PROGRESSION. THIS STUDY AIMED TO INVESTIGATE LONG NON-CODING RNA (LNCRNA) N6-METHYLADENOSINE (M(6)A) MODIFICATION AND ITS ROLE IN REGULATING RIF PROGRESSION. METHODS: UNILATERAL URETERAL OCCLUSION (UUO) WAS EMPLOYED TO CONSTRUCT THE RIF IN VIVO MODEL; AND TGF-BETA1-TREATED HK-2 AND HKC-8 CELLS WERE USED FOR IN VITRO EXPERIMENTS. THE MRNA AND PROTEIN EXPRESSIONS WERE ASSESSED USING QRT-PCR AND WESTERN BLOT. THE PROLIFERATION AND MIGRATION WERE EVALUATED BY EDU ASSAY AND TRANSWELL ASSAY, RESPECTIVELY. IN ADDITION, LEVELS OF INFLAMMATORY CYTOKINES WERE DETERMINED BY ELISA ASSAY AND QRT-PCR. MOREOVER, LNCRNA GAS5 M(6)A LEVEL WAS DETECTED USING ME-RIP ASSAY. HE AND MASSON STAINING WERE EMPLOYED TO EVALUATE FIBROTIC LESIONS OF THE KIDNEY. RESULTS: FTO EXPRESSION WAS ELEVATED IN HK-2 AND HKC-8 CELLS AFTER TGF-BETA1 TREATMENT AND MOUSE KIDNEY TISSUE FOLLOWING UUO, AND LNCRNA GAS5 WAS DOWNREGULATED. LNCRNA GAS5 OVEREXPRESSION OR FTO SILENCING SUPPRESSED TGF-BETA1-INDUCED THE INCREASE OF EMT-RELATED PROTEINS (VIMENTIN, SNAIL AND N-CADHERIN) AND INFLAMMATORY CYTOKINES (IL-6, IL-1BETA AND TNF-ALPHA) LEVELS IN HK-2 CELLS. FTO SUPPRESSED LNCRNA GAS5 EXPRESSION BY REDUCING THE M6A MODIFICATION OF LNCRNA GAS5. ADDITIONALLY, FTO KNOCKDOWN COULD SUPPRESS EMT PROCESS AND INFLAMMATION RESPONSE INDUCED BY TGF-BETA1 AND UUO IN VITRO AND IN VIVO. AS EXPECTED, FTO KNOCKDOWN ABROGATED THE PROMOTION EFFECTS OF LNCRNA GAS5 SILENCING ON TGF-BETA1-INDUCED EMT PROCESS AND INFLAMMATION RESPONSE IN HK-2 AND HKC-8 CELLS. CONCLUSION: FTO PROMOTED EMT PROCESS AND INFLAMMATION RESPONSE THROUGH REDUCING THE M(6)A MODIFICATION OF LNCRNA GAS5. 2022