1 3264 142 HEPATITIS VIRUS INFECTION AFFECTS DNA METHYLATION IN MICE WITH HUMANIZED LIVERS. BACKGROUND & AIMS: CELLS OF TUMORS ASSOCIATED WITH CHRONIC INFLAMMATION FREQUENTLY HAVE ALTERED PATTERNS OF DNA METHYLATION, INCLUDING HEPATOCELLULAR CARCINOMAS. CHRONIC HEPATITIS HAS ALSO BEEN ASSOCIATED WITH ABERRANT DNA METHYLATION, BUT LITTLE IS KNOWN ABOUT THEIR RELATIONSHIP. METHODS: PYROSEQUENCING WAS USED TO DETERMINE THE METHYLATION STATUS OF CULTURED HUH7.5.1 HEPATOMA CELLS AFTER HEPATITIS C VIRUS (HCV) INFECTION. WE ALSO STUDIED MICE WITH SEVERE COMBINED IMMUNODEFICIENCY CARRYING THE UROKINASE-TYPE PLASMINOGEN ACTIVATOR TRANSGENE CONTROLLED BY AN ALBUMIN PROMOTER (UROKINASE-TYPE PLASMINOGEN ACTIVATOR/SEVERE COMBINED IMMUNODEFICIENT MICE), IN WHICH UP TO 85% OF HEPATOCYTES WERE REPLACED BY HUMAN HEPATOCYTES (CHIMERIC MICE). MICE WERE GIVEN INTRAVENOUS INJECTIONS OF HEPATITIS B VIRUS (HBV) OR HCV, LIVER TISSUES WERE COLLECTED, AND DNA METHYLATION PROFILES WERE DETERMINED AT DIFFERENT TIME POINTS AFTER INFECTION. WE ALSO COMPARED METHYLATION PATTERNS BETWEEN PAIRED SAMPLES OF HEPATOCELLULAR CARCINOMAS AND ADJACENT NONTUMOR LIVER TISSUES FROM PATIENTS. RESULTS: NO REPRODUCIBLE CHANGES IN DNA METHYLATION WERE OBSERVED AFTER INFECTION OF HUH7.5.1 CELLS WITH HCV. LIVERS FROM HBV- AND HCV-INFECTED MICE HAD GENOME-WIDE, TIME-DEPENDENT CHANGES IN DNA METHYLATION, COMPARED WITH UNINFECTED UROKINASE-TYPE PLASMINOGEN ACTIVATOR/SEVERE COMBINED IMMUNODEFICIENT MICE. THERE WERE CHANGES IN 160 +/- 63 GENES IN HBV-INFECTED AND 237 +/- 110 GENES IN HCV-INFECTED MICE. METHYLATION OF 149 COMMON GENES INCREASED IN HBV- AND HCV-INFECTED MICE; METHYLATION OF SOME OF THESE GENES ALSO INCREASED IN HEPATOCELLULAR CARCINOMA SAMPLES FROM PATIENTS COMPARED WITH NONTUMOR TISSUES. EXPRESSION OF IFNG, WHICH IS EXPRESSED BY NATURAL KILLER CELLS, INCREASED SIGNIFICANTLY IN CHIMERIC LIVERS, IN CONCORDANCE WITH INDUCTION OF DNA METHYLATION, AFTER INFECTION WITH HBV OR HCV. INDUCTION OF IFNG WAS REDUCED AFTER ADMINISTRATION OF AN INHIBITOR OF NATURAL KILLER CELL FUNCTION (ANTI-ASIALO GM1). CONCLUSIONS: IN CHIMERIC MICE WITH HUMANIZED LIVERS, INFECTION WITH HBV AND HCV APPEARS TO ACTIVATE A NATURAL KILL CELL-DEPENDENT INNATE IMMUNE RESPONSE. THIS CONTRIBUTES TO THE INDUCTION AND ACCUMULATION OF ABERRANT DNA METHYLATION IN HUMAN HEPATOCYTES.	2014	
                                                                                                                                                                                                                                                                                                                                                                                                                                                         
2 3646  34 INCREASED PROTEIN EXPRESSION OF DNA METHYLTRANSFERASE (DNMT) 1 IS SIGNIFICANTLY CORRELATED WITH THE MALIGNANT POTENTIAL AND POOR PROGNOSIS OF HUMAN HEPATOCELLULAR CARCINOMAS. ALTERATION OF DNA METHYLATION IS ONE OF THE MOST CONSISTENT EPIGENETIC CHANGES IN HUMAN CANCERS. DNA METHYLTRANSFERASE (DNMT) 1 IS A MAJOR ENZYME INVOLVED IN ESTABLISHING GENOMIC METHYLATION PATTERNS. MOST OF THE STUDIES CONCERNING DNMT1 EXPRESSION IN HUMAN CANCERS HAVE BEEN PERFORMED ONLY AT THE MRNA LEVEL. TO DIRECTLY EXAMINE DNMT1 PROTEIN EXPRESSION LEVELS DURING HUMAN HEPATOCARCINOGENESIS, 16 HISTOLOGICALLY NORMAL LIVER TISSUES, 51 NONCANCEROUS LIVER TISSUES EXHIBITING CHRONIC HEPATITIS OR CIRRHOSIS, WHICH ARE CONSIDERED TO BE PRECANCEROUS CONDITIONS, AND 53 HEPATOCELLULAR CARCINOMAS (HCCS) WERE SUBJECTED TO IMMUNOHISTOCHEMIC EXAMINATION. IF MORE THAN 20% OF THE CELLS EXHIBITED NUCLEAR DNMT1 STAINING, THE TISSUE SAMPLE WAS CONSIDERED TO BE DNMT1-POSITIVE. DNMT1 IMMUNOREACTIVITY WAS OBSERVED IN 23 (43%) OF THE HCCS, BUT IN NONE (0%) OF THE HISTOLOGICALLY NORMAL LIVER OR NONCANCEROUS LIVER TISSUES EXHIBITING CHRONIC HEPATITIS OR CIRRHOSIS. THE INCIDENCE OF INCREASED DNMT1 PROTEIN EXPRESSION IN HCCS CORRELATED SIGNIFICANTLY WITH POOR TUMOR DIFFERENTIATION (P = 0.0006) AND PORTAL VEIN INVOLVEMENT (P = 0.0002). MOREOVER, THE RECURRENCE-FREE (P = 0.0001) AND OVERALL (P < 0.0001) SURVIVAL RATES OF PATIENTS WITH HCCS EXHIBITING INCREASED DNMT1 PROTEIN EXPRESSION WERE SIGNIFICANTLY LOWER THAN THOSE OF PATIENTS WITH HCCS THAT DID NOT EXHIBIT INCREASED EXPRESSION. INCREASED DNMT1 PROTEIN EXPRESSION MAY PLAY A CRITICAL ROLE IN THE MALIGNANT PROGRESSION OF HCCS AND BE A BIOLOGIC PREDICTOR OF BOTH HCC RECURRENCE AND A POOR PROGNOSIS IN HCC PATIENTS.	2003	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          
3 3248  40 HEPATITIS B VIRUS GENOME ASYMMETRY IN HEPATOCELLULAR CARCINOMA. BACKGROUND: THE ASSOCIATION BETWEEN HEPATITIS B VIRUS (HBV) MUTATIONS AND HEPATOCARCINOGENESIS WERE REPORTED IN THE LITERATURE. PREFERENCE FOR G OVER C IN THE LEADING DNA STRAND HAS BEEN REPORTED TO ACCOUNT FOR THE ASYMMETRY IN NUCLEOTIDE (NT) COMPOSITION. THE AIM OF THIS STUDY WAS TO ANALYZE THE COMPLETE GENOME SEQUENCE AND COMPOSITIONAL ASYMMETRY OF HBV IN DIFFERENT STAGES OF HEPATITIS B. METHODS: FULL GENOME SEQUENCING OF 24 PATIENTS WITH CHRONIC HEPATITIS B, SOME OF WHOM ALSO HAD CIRRHOSIS AND HEPATOCELLULAR CARCINOMA (HCC) WAS PERFORMED. MUTATIONS ANALYSIS WAS IMPLEMENTED IN A COMPARISON WITH A HBV GENOTYPE D REFERENCE FROM AN INTERNATIONAL DNA DATABASE. CPGPROD, A WEB-BASED APPLICATION, WAS USED TO EVALUATE CG CONTENT AND PREDICT CPG ISLANDS. RESULTS: ALL STRAINS WERE 3182 BASE PAIRS (BP) IN LENGTH, EXCEPT FOR TWO CASES OF HCC IN WHICH 9 AND 21 NT, RESPECTIVELY, WERE DELETED IN PRES2. THE GENETIC RELATEDNESS OF THESE ISOLATES WAS 97%-100%. THERE WERE COMMON CPG-RICH REGIONS IN ALL 24 ISOLATED FULL GENOME SEQUENCES, HOWEVER A STRONG NEGATIVE GC SKEW FOR FORMING A CPG ISLAND IN THE MINUS STRAND WERE EXHIBITED IN OVERLAP WITH ENHANCER I IN THREE HCC PATIENTS, A CIRRHOTIC PATIENT AND THREE WITH CHRONIC HEPATITIS. CONCLUSION: THE HIGH PERCENTAGE OF SEQUENCE IDENTITY BETWEEN HBV ISOLATES IN OUR PATIENTS DEMONSTRATES THAT GENOMIC FACTORS, EXCEPT FOR GENOTYPE, ARE INVOLVED IN HEPATOCARCINOGENESIS. VARIATIONS IN GC CONTENT WHICH WERE CAUSED BY A DIFFERENT SPECTRUM OF MUTATIONS MAY AFFECT DNA COMPOSITIONAL ASYMMETRY AND EPIGENETIC MODIFICATION OF HBV DNA IN HCC.	2012	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      
4 6692  40 VARIABLE DNA METHYLATION PATTERNS ASSOCIATED WITH PROGRESSION OF DISEASE IN HEPATOCELLULAR CARCINOMAS. HEPATOCELLULAR CARCINOMA (HCC) MOST COMMONLY ARISES FROM CHRONIC INFLAMMATION DUE TO VIRAL INFECTION, AS A RESULT OF GENETIC AND EPIGENETIC ABNORMALITIES. A GLOBAL PICTURE OF EPIGENETIC CHANGES IN HCC IS LACKING. WE USED METHYLATED CPG ISLAND AMPLIFICATION MICROARRAYS (MCAMS) TO STUDY 6458 CPG ISLANDS IN HCC AND ADJACENT PRENEOPLASTIC TISSUES [CHRONIC HEPATITIS (CH) OR LIVER CIRRHOSIS (LC)] IN COMPARISON WITH NORMAL LIVER TISSUES WHERE NEITHER VIRAL INFECTION NOR HEPATITIS HAS EXISTED. MCAM IDENTIFIED 719 (11%) PROMINENT GENES OF HYPERMETHYLATION IN HCCS. HCCS ARISING FROM LC HAD SIGNIFICANTLY MORE METHYLATION THAN THOSE ARISING FROM CH (1249 GENES OR 19% VERSUS 444 GENES OR 7%, P < 0.05). THERE WERE FOUR PATTERNS OF ABERRANT METHYLATION: TYPE I (4%, E.G. MATRIX METALLOPROTEINASE 14) SHOWS A SUBSTANTIALLY HIGH METHYLATION LEVEL IN ADJACENT TISSUE AND DOES NOT INCREASE FURTHER IN CANCER. TYPE II (55%, E.G. RASSF1A) SHOWS PROGRESSIVELY INCREASING METHYLATION FROM ADJACENT TISSUE TO HCC. TYPE III (4%, E.G. GNA14) SHOWS DECREASED METHYLATION IN ADJACENT TISSUE BUT EITHER SIMILAR OR INCREASED METHYLATION IN HCC. TYPE IV (37%, E.G. CDKN2A) SHOWS LOW LEVELS OF METHYLATION IN NORMAL TISSUE AND ADJACENT TISSUE BUT HIGH LEVELS IN HCC. THESE DNA METHYLATION CHANGES WERE CONFIRMED BY QUANTITATIVE PYROSEQUENCING METHYLATION ANALYSIS IN REPRESENTATIVE 24 GENES AND WERE ANALYZED FOR CORRELATION WITH CLINICOPATHOLOGICAL PARAMETERS IN 38 PATIENTS. INTRIGUINGLY, METHYLATION IN THE TYPE IV GENES IS CHARACTERISTIC OF MODERATELY/POORLY DIFFERENTIATED CANCER. OUR GLOBAL EPIGENOME ANALYSIS REVEALS DISTINCT PATTERNS OF METHYLATION THAT ARE PROBABLY TO REPRESENT DIFFERENT PATHOPHYSIOLOGIC PROCESSES IN HCCS.	2008	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
5 3190  51 HCV-INDUCED EPIGENETIC CHANGES ASSOCIATED WITH LIVER CANCER RISK PERSIST AFTER SUSTAINED VIROLOGIC RESPONSE. BACKGROUND & AIMS: CHRONIC HEPATITIS C VIRUS (HCV) INFECTION IS AN IMPORTANT RISK FACTOR FOR HEPATOCELLULAR CARCINOMA (HCC). DESPITE EFFECTIVE ANTIVIRAL THERAPIES, THE RISK FOR HCC IS DECREASED BUT NOT ELIMINATED AFTER A SUSTAINED VIROLOGIC RESPONSE (SVR) TO DIRECT-ACTING ANTIVIRAL (DAA) AGENTS, AND THE RISK IS HIGHER IN PATIENTS WITH ADVANCED FIBROSIS. WE INVESTIGATED HCV-INDUCED EPIGENETIC ALTERATIONS THAT MIGHT AFFECT RISK FOR HCC AFTER DAA TREATMENT IN PATIENTS AND MICE WITH HUMANIZED LIVERS. METHODS: WE PERFORMED GENOME-WIDE CHIPMENTATION-BASED CHIP-SEQ AND RNA-SEQ ANALYSES OF LIVER TISSUES FROM 6 PATIENTS WITHOUT HCV INFECTION (CONTROLS), 18 PATIENTS WITH CHRONIC HCV INFECTION, 8 PATIENTS WITH CHRONIC HCV INFECTION CURED BY DAA TREATMENT, 13 PATIENTS WITH CHRONIC HCV INFECTION CURED BY INTERFERON THERAPY, 4 PATIENTS WITH CHRONIC HEPATITIS B VIRUS INFECTION, AND 7 PATIENTS WITH NONALCOHOLIC STEATOHEPATITIS IN EUROPE AND JAPAN. HCV-INDUCED EPIGENETIC MODIFICATIONS WERE MAPPED BY COMPARATIVE ANALYSES WITH MODIFICATIONS ASSOCIATED WITH OTHER LIVER DISEASE ETIOLOGIES. UPA/SCID MICE WERE ENGRAFTED WITH HUMAN HEPATOCYTES TO CREATE MICE WITH HUMANIZED LIVERS AND GIVEN INJECTIONS OF HCV-INFECTED SERUM SAMPLES FROM PATIENTS; MICE WERE GIVEN DAAS TO ERADICATE THE VIRUS. PATHWAYS ASSOCIATED WITH HCC RISK WERE IDENTIFIED BY INTEGRATIVE PATHWAY ANALYSES AND VALIDATED IN ANALYSES OF PAIRED HCC TISSUES FROM 8 PATIENTS WITH AN SVR TO DAA TREATMENT OF HCV INFECTION. RESULTS: WE FOUND CHRONIC HCV INFECTION TO INDUCE SPECIFIC GENOME-WIDE CHANGES IN H3K27AC, WHICH CORRELATED WITH CHANGES IN EXPRESSION OF MRNAS AND PROTEINS. THESE CHANGES PERSISTED AFTER AN SVR TO DAAS OR INTERFERON-BASED THERAPIES. INTEGRATIVE PATHWAY ANALYSES OF LIVER TISSUES FROM PATIENTS AND MICE WITH HUMANIZED LIVERS DEMONSTRATED THAT HCV-INDUCED EPIGENETIC ALTERATIONS WERE ASSOCIATED WITH LIVER CANCER RISK. COMPUTATIONAL ANALYSES ASSOCIATED INCREASED EXPRESSION OF SPHK1 WITH HCC RISK. WE VALIDATED THESE FINDINGS IN AN INDEPENDENT COHORT OF PATIENTS WITH HCV-RELATED CIRRHOSIS (N = 216), A SUBSET OF WHICH (N = 21) ACHIEVED VIRAL CLEARANCE. CONCLUSIONS: IN AN ANALYSIS OF LIVER TISSUES FROM PATIENTS WITH AND WITHOUT AN SVR TO DAA THERAPY, WE IDENTIFIED EPIGENETIC AND GENE EXPRESSION ALTERATIONS ASSOCIATED WITH RISK FOR HCC. THESE ALTERATIONS MIGHT BE TARGETED TO PREVENT LIVER CANCER IN PATIENTS TREATED FOR HCV INFECTION.	2019	
                                                                                                                                                                                                                                                         
6   42  48 A COMPREHENSIVE GENOME-WIDE PROFILING COMPARISON BETWEEN HBV AND HCV INFECTED HEPATOCELLULAR CARCINOMA. BACKGROUND: HEPATOCELLULAR CARCINOMA (HCC) IS ONE OF THE MOST COMMON CANCERS WORLDWIDE, ESPECIALLY IN EAST ASIA. EVEN WITH THE PROGRESS IN THERAPY, 5-YEAR SURVIVAL RATES REMAIN UNSATISFIED. CHRONIC INFECTION WITH THE HEPATITIS B VIRUS (HBV) OR HEPATITIS C VIRUS (HCV) HAS BEEN EPIDEMIOLOGICALLY ASSOCIATED WITH HCC AND IS THE MAJOR ETIOLOGY IN THE EAST ASIAN POPULATION. THE DETAILED MECHANISM, ESPECIALLY THE CHANGES OF DNA METHYLATION AND GENE EXPRESSION BETWEEN THE TWO TYPES OF VIRUS-RELATED HCC, AND THEIR CONTRIBUTIONS TO THE HCC DEVELOPMENT, METASTASIS, AND RECURRENCE REMAIN LARGELY UNKNOWN. METHODS: IN THIS INTEGRATED ANALYSIS, WE CHARACTERIZED GENOME-SCALE PROFILES OF HBV AND HCV INFECTED HCC BY COMPARING THEIR GENE EXPRESSION PATTERN, METHYLATION PROFILES, AND COPY NUMBER VARIATIONS FROM THE PUBLICLY ACCESSIBLE DATA OF THE CANCER GENOME ATLAS PROGRAM (TCGA). RESULTS: THE HLA-A, STAT1, AND OAS2 GENES WERE HIGHLY ENRICHED AND UP-REGULATED DISCOVERED IN THE HCV-INFECTED HCC. HYPOMETHYLATION BUT NOT COPY NUMBER VARIATIONS MIGHT BE THE MAJOR FACTOR FOR THE UP-REGULATION OF THESE IMMUNE-RELATED GENES IN HCV-INFECTED HCC. CONCLUSIONS: THE RESULTS INDICATED THE DIFFERENT EPIGENETIC CHANGES OF HBV/HCV RELATED HEPATOCARCINOGENESIS. THE TOP UP-REGULATED GENES IN HCV GROUP WERE SIGNIFICANTLY CLUSTERED IN THE IMMUNE-RELATED AND DEFENSE RESPONSE PATHWAYS. THESE FINDINGS WILL HELP US TO UNDERSTAND THE PATHOGENESIS OF HBV/HCV ASSOCIATED HEPATOCELLULAR CARCINOMA.	2019	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            
7 2682  34 EVALUATION OF SERUM LINE-1 HYPOMETHYLATION AS A PROGNOSTIC MARKER FOR HEPATOCELLULAR CARCINOMA. BACKGROUND AND STUDY AIMS: GLOBAL HYPOMETHYLATION IS ONE OF THE MOST CONSISTENT EPIGENETIC CHANGES IN CANCER. DEVELOPMENT OF HEPATOCELLULAR CARCINOMA (HCC) MUST BE UNDERSTOOD AS A MULTISTEP PROCESS WITH ACCUMULATION OF GENETIC AND EPIGENETIC ALTERATIONS. IN THE LAST DECADES, IN ADDITION TO GENETIC ALTERATIONS, EPIGENETIC CHANGES HAVE BEEN RECOGNIZED AS AN IMPORTANT AND ALTERNATIVE MECHANISM IN TUMOURIGENESIS. WE INVESTIGATED THE CLINICAL IMPLICATIONS OF GLOBAL HYPOMETHYLATION IN THE SERA OF PATIENTS WITH HEPATOCELLULAR CARCINOMA (HCC). PATIENTS AND METHODS: PCR WAS USED TO ASSESS THE METHYLATION STATUS OF LONG INTERSPERSED NUCLEAR ELEMENT TYPE 1 (LINE-1) REPETITIVE SEQUENCES IN GENOMIC DNA DERIVED FROM SERA OF 50 PATIENTS WITH HCC, 20 PATIENTS WITH CIRRHOSIS, 20 PATIENTS WITH CHRONIC HEPATITIS C AND 10 HEALTHY SUBJECTS. RESULTS: SERUM GENOME HYPOMETHYLATION WAS SIGNIFICANTLY INCREASED IN PATIENTS WITH HCC (P<0.001). THE LEVELS OF SERUM LINE-1 HYPOMETHYLATION AT INITIAL PRESENTATION CORRELATED SIGNIFICANTLY WITH TUMOUR SIZE, TUMOUR NUMBER AND ALPHA-FOETOPROTEIN LEVEL. MOREOVER HIGH SERUM LINE-1 HYPOMETHYLATION CORRELATES SIGNIFICANTLY WITH POOR SURVIVAL. CONCLUSION: SERUM LINE-1 HYPOMETHYLATION MAY SERVE AS A PROGNOSTIC MARKER FOR PATIENTS WITH HCC.	2011	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
8 1617  39 DNA METHYLTRANSFERASE EXPRESSION AND DNA METHYLATION IN HUMAN HEPATOCELLULAR CARCINOMA AND THEIR CLINICOPATHOLOGICAL CORRELATION. ABERRANT DNA METHYLATION ON CPG ISLANDS IS ONE OF THE MOST CONSISTENT EPIGENETIC CHANGES IN HUMAN CANCERS, AND THE METHYLATION PROCESS IS CATALYZED BY DNA METHYLTRANSFERASE (DNMT). WE EVALUATED I) THE MRNA LEVELS OF THREE DNMTS; DNMT1, DNMT3A AND DNMT3B, IN 25 HEPATOCELLULAR CARCINOMAS (HCCS), IN THEIR CORRESPONDING NON-CANCEROUS LIVER TISSUES AND IN 7 NORMAL LIVERS BY USING REAL-TIME REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION; II) NUCLEAR EXPRESSION OF DNMT1 AND DNMT3A PROTEINS IN THE HCCS BY IMMUNOHISTOCHEMISTRY, III) THE METHYLATION STATUS OF 5 GENES; P16, P15, E-CADHERIN, HIC-1 AND RASSF1A IN THE SAME TISSUES, AND IV) THE RELATIONSHIPS BETWEEN THE ABOVE RESULTS AND THE CLINICOPATHOLOGICAL CHARACTERISTICS, INCLUDING PROGNOSIS. THE DIFFERENCES IN MRNA EXPRESSION LEVELS FOR DNMT1, DNMT3A AND DNMT3B WERE STATISTICALLY SIGNIFICANT BETWEEN HCC AND NORMAL LIVERS (P<0.001), HCC AND CHRONIC HEPATITIS (P<0.001) AND HCC AND CIRRHOSIS (P<0.001). AN INCREASE IN MRNA EXPRESSION LEVELS OF >4-FOLD FOR DNMT3B IN HCCS WAS SIGNIFICANTLY ASSOCIATED WITH A POORER OVERALL SURVIVAL (P=0.027) AND SHORTER METASTASIS-FREE SURVIVAL (P=0.0299). A POORER RECURRENCE-FREE SURVIVAL WAS NOTED IN HCCS WITH A >4-FOLD INCREASE IN DNMT3A MRNA (P=0.0120). THE AVERAGE NUMBERS OF METHYLATED GENES WERE 0, 1.27, 1.38 AND 2.72 FOR NORMAL LIVERS, CHRONIC HEPATITIS, CIRRHOSIS AND HCCS, RESPECTIVELY, AND THIS PROGRESSIVE INCREASE FROM NORMAL LIVERS TO CHRONIC HEPATITIS/CIRRHOSIS THROUGH HCC MAY SUGGEST THAT TUMOR SUPPRESSOR GENE METHYLATION IS AN EARLY EVENT IN HEPATOCARCINOGENESIS. THESE RESULTS FIRST SUGGEST THAT HEPATOCARCINOGENESIS INVOLVES AN INCREASED EXPRESSION OF DNMT1, DNMT3A AND DNMT3B MRNA AND A PROGRESSIVE INCREASE IN THE NUMBER OF METHYLATED GENES FROM NORMAL LIVER, CHRONIC HEPATITIS/CIRRHOSIS TO HCC AND SECONDLY THAT AN INCREASE IN THE DNMT3A AND DNMT3B MRNA LEVELS IN HCCS RELATIVE TO THEIR NON-CANCEROUS TISSUES MAY BE A PREDICTOR OF POOR SURVIVAL.	2007	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             
9 2234  34 EPIGENETIC MODIFICATIONS PRECEDE MOLECULAR ALTERATIONS AND DRIVE HUMAN HEPATOCARCINOGENESIS. DEVELOPMENT OF PRIMARY LIVER CANCER IS A MULTISTAGE PROCESS. DETAILED UNDERSTANDING OF SEQUENTIAL EPIGENETIC ALTERATIONS IS LARGELY MISSING. HERE, WE PERFORMED INFINIUM HUMAN METHYLATION 450K BEADCHIPS AND RNA-SEQ ANALYSES FOR GENOME-WIDE METHYLOME AND TRANSCRIPTOME PROFILING OF CIRRHOTIC LIVER (N = 7), LOW- (N = 4) AND HIGH-GRADE (N = 9) DYSPLASTIC LESIONS, AND EARLY (N = 5) AND PROGRESSED (N = 3) HEPATOCELLULAR CARCINOMAS (HCC) SYNCHRONOUSLY DETECTED IN 8 PATIENTS WITH HCC WITH CHRONIC HEPATITIS B INFECTION. INTEGRATIVE ANALYSES OF EPIGENETICALLY DRIVEN MOLECULAR CHANGES WERE IDENTIFIED AND VALIDATED IN 2 INDEPENDENT COHORTS COMPRISING 887 HCCS. MITOCHONDRIAL DNA SEQUENCING WAS FURTHER EMPLOYED FOR CLONALITY ANALYSES, INDICATING MULTICLONAL ORIGIN IN THE MAJORITY OF INVESTIGATED HCCS. ALTERATIONS IN DNA METHYLATION PROGRESSIVELY INCREASED FROM LIVER CIRRHOSIS (CL) TO DYSPLASTIC LESIONS AND REACHED A MAXIMUM IN EARLY HCCS. ASSOCIATED EARLY ALTERATIONS IDENTIFIED BY INGENUITY PATHWAY ANALYSIS (IPA) INVOLVED APOPTOSIS, IMMUNE REGULATION, AND STEMNESS PATHWAYS, WHILE LATE CHANGES CENTERED ON CELL SURVIVAL, PROLIFERATION, AND INVASION. WE FURTHER VALIDATED 23 PUTATIVE EPIDRIVERS WITH CONCOMITANT EXPRESSION CHANGES AND ASSOCIATED WITH OVERALL SURVIVAL. FUNCTIONALLY, STRIATIN 4 (STRN4) WAS DEMONSTRATED TO BE EPIGENETICALLY REGULATED, AND INHIBITION OF STRN4 SIGNIFICANTLY SUPPRESSED TUMORIGENICITY OF HCC CELL LINES. OVERALL, APPLICATION OF INTEGRATIVE GENOMIC ANALYSES DEFINES EPIGENETIC DRIVER ALTERATIONS AND PROVIDES PROMISING TARGETS FOR POTENTIALLY NOVEL THERAPEUTIC APPROACHES.	2021	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
10 3897  42 LARGE-SCALE ANALYSIS OF THE GENETIC AND EPIGENETIC ALTERATIONS IN HEPATOCELLULAR CARCINOMA FROM SOUTHEAST CHINA. OUR KNOWLEDGE ABOUT MOLECULAR ALTERATIONS DURING HEPATOCARCINOGENESIS IS STILL FRAGMENTARY, DUE TO LACK OF COMPREHENSIVE GENETIC AND EPIGENETIC ANALYSES IN THE SAME SET OF HEPATOCELLULAR CARCINOMAS (HCCS). IN THIS STUDY, WE CONDUCTED A LARGE-SCALE ANALYSIS, INCLUDING MUTATION SCREENING IN 50 GENES AND METHYLATION ASSAYS IN THREE GENES IN 54 PAIRS OF HCCS AND THEIR NEIGHBORING NON-CANCEROUS TISSUES. ALL SAMPLES WERE COLLECTED FROM THE RESIDENTS IN SOUTHEAST CHINA. WE FOUND HBV INFECTION AND CHRONIC HEPATITIS/CIRRHOSIS IN 83.3% AND 98.1% OF THE CASES, RESPECTIVELY. MUTATIONS WERE IDENTIFIED IN 18 OUT OF 54 (33.3%) SAMPLES, WITH P53 ALTERATIONS IN 14 CASES AND BETA-CATENIN MUTATIONS IN FOUR TUMORS. NO MUTATIONS WERE IDENTIFIED IN THE NEIGHBORING TISSUES. INTERESTINGLY, 9 OUT OF 14 (64.3%) TUMORS CARRYING P53 MUTATIONS DISPLAYED SUBSTITUTION OF SERINE BY ARGININE AT CODON 249, A CHARACTERISTIC CHANGE BELIEVED TO BE INDUCED BY AFLATOXIN-B1. FURTHERMORE, P53 MUTATION WAS SIGNIFICANTLY ASSOCIATED WITH SHORTER RECURRENCE-FREE SURVIVAL (P=0.004). THE RESULTS ALSO REVEALED ABERRANT METHYLATION IN TWO OR MORE GENES IN AS HIGH AS 90% OF TUMORS AND 40% OF ADJACENT TISSUES. THE FREQUENCY OF RASSF1A HYPERMETHYLATION WAS MUCH HIGHER THAN THAT OF P16INK4A AND HAI2 IN BOTH HCC AND NEIGHBORING TISSUES, INDICATING THAT DEREGULATION OF RASSF1A MAY PRECEDE THE OTHER TWO GENES. THESE DATA SUGGEST THAT ABERRANT METHYLATION OCCURS BEFORE MUTATION AND IS AN EARLY EVENT IN THE DEVELOPMENT OF THIS SET OF HCC. OUR FINDINGS HIGHLIGHT P53 AS A PROGNOSTIC FACTOR OF HCC AND RASSF1A AS A POTENTIAL TARGET IN PREVENTING MALIGNANT TRANSFORMATION OF HEPATOCYTES.	2008	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
11 3825  33 INVESTIGATION OF CTNNB1 GENE MUTATIONS AND EXPRESSION IN HEPATOCELLULAR CARCINOMA AND CIRRHOSIS IN ASSOCIATION WITH HEPATITIS B VIRUS INFECTION. HEPATITIS B VIRUS (HBV), ALONG WITH HEPATITIS C VIRUS CHRONIC INFECTION, REPRESENTS A MAJOR RISK FACTOR FOR HEPATOCELLULAR CARCINOMA (HCC) DEVELOPMENT. HOWEVER, MOLECULAR MECHANISMS INVOLVED IN THE DEVELOPMENT OF HCC ARE NOT YET COMPLETELY UNDERSTOOD. RECENT STUDIES HAVE INDICATED THAT MUTATIONS IN CTNNB1 GENE ENCODING FOR BETA-CATENIN PROTEIN LEAD TO ABERRANT ACTIVATION OF THE WNT/ BETA-CATENIN PATHWAY. THE MUTATIONS IN TURN ACTIVATE SEVERAL DOWNSTREAM GENES, INCLUDING C-MYC, PROMOTING THE NEOPLASTIC PROCESS. THE PRESENT STUDY EVALUATED THE MUTATIONAL PROFILE OF THE CTNNB1 GENE AND EXPRESSION LEVELS OF CTNNB1 AND C-MYC GENES IN HBV-RELATED HCC, AS WELL AS IN CIRRHOTIC AND CONTROL TISSUES. MUTATIONAL ANALYSIS OF THE BETA-CATENIN GENE AND HBV GENOTYPING WERE CONDUCTED BY DIRECT SEQUENCING. EXPRESSION OF BETA-CATENIN AND C-MYC GENES WAS ASSESSED USING REAL-TIME PCR. AMONG THE HCC CASES, 18.1% SHOWED MISSENSE POINT MUTATION IN EXON 3 OF CTNNB1, MORE FREQUENTLY IN CODONS 32, 33, 38 AND 45. THE FREQUENCY OF MUTATION IN THE HOTSPOTS OF EXON 3 WAS SIGNIFICANTLY HIGHER IN NON-VIRAL HCCS (29.4%) RATHER THAN HBV-RELATED CASES (12.7%, P = 0.021). THE EXPRESSION OF BETA-CATENIN AND C-MYC GENES WAS FOUND UPREGULATED IN CIRRHOTIC TISSUES IN ASSOCIATION WITH HBV INFECTION. MUTATIONS AT BOTH PHOSPHORYLATION AND NEIGHBORING SITES WERE ASSOCIATED WITH INCREASED ACTIVITY OF THE WNT PATHWAY. THE RESULTS DEMONSTRATED THAT MUTATED BETA-CATENIN CAUSED ACTIVATION OF THE WNT PATHWAY, BUT THE RATE OF CTNNB1 GENE MUTATIONS WAS NOT RELATED TO HBV INFECTION. HBV FACTORS MAY DEREGULATE THE WNT PATHWAY BY CAUSING EPIGENETIC ALTERATIONS IN THE HBV-RELATED HCC.	2020	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            
12 5844  37 STUDY OF PROMOTER HYPOMETHYLATION PROFILES OF RAS ONCOGENES IN HEPATOCELLULAR CARCINOMA DERIVED FROM HEPATITIS C VIRUS GENOTYPE 3A IN PAKISTANI POPULATION. EPIGENETIC MODIFICATIONS SUCH AS DNA METHYLATION CONTRIBUTE TO PROGRESSION OF HEPATITIS C VIRUS (HCV) INFECTION TO LIFE-THREATENING HEPATOCELLULAR CARCINOMA (HCC) BY PROMOTING THE SILENCING OF TUMOR SUPPRESSOR GENES THROUGH DNA HYPERMETHYLATION AND BY CAUSING GENOMIC INSTABILITY THROUGH GLOBAL HYPOMETHYLATION. HOWEVER FEW STUDIES HAVE ADDRESSED THE PROMOTER REGION HYPOMETHYLATION STATUS OF THE ONCOGENES INVOLVED IN HCV DERIVED HCC. IN THIS STUDY, WE ANALYZED THE PROMOTER REGION METHYLATION PATTERN OF RAS ONCOGENES (HRAS, KRAS, AND NRAS) USING METHYLATION-SPECIFIC PCR FOR 50 CHRONIC HCV PATIENTS INFECTED WITH GENOTYPE 3A (27 HCC PATIENTS AND 23 CONTROL NON-HCC PATIENTS). METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION ANALYSIS REVEALED THAT THE NRAS ONCOGENE PROMOTER (P = .0025) WAS SIGNIFICANTLY HYPOMETHYLATED IN HCC PATIENTS COMPARED TO THE NON-HCC PATIENTS SUGGESTING ITS CONTRIBUTION TO THE PROGRESSION OF HCV TOWARDS HCC. TO IDENTIFY THE AGENT FOR ALTERATION IN THE RAS ONCOGENE EXPRESSION, 7 HCV GENES WERE EXPRESSED IN THE HUH-7 CELL LINE FOLLOWED BY MEASUREMENT OF THE NRAS EXPRESSION LEVEL IN HUH-7 BY A QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION. AN INCREASE IN THE MESSENGER RNA LEVEL OF THE NRAS GENE WAS DETECTED WHEN HUH-7 WERE TRANSFECTED WITH CORE, NS5A, AND NS2 GENES. OUR FINDINGS SUGGEST THE INVOLVEMENT OF NRAS ONCOGENE IN THE PATHOGENESIS OF HCV3A DERIVED HCC IN PAKISTANI POPULATION AND ALSO IDENTIFIES THE HCV GENES RESPONSIBLE FOR ITS ENHANCED EXPRESSION. OUR STUDY RAISES THE HYPOTHESIS THAT A SINGLE HCV GENE MAY INCREASE THE CHANCES OF MALIGNANCY. THEREFORE, OUR STUDY MAY HAVE IDENTIFIED A USEFUL EPIGENETIC BIOMARKER OF HCC PROGRESSION IN HCV PATIENTS AND MAY HELP TO DEVELOP NOVEL DIAGNOSTIC TOOLS.	2018	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
13 1615  29 DNA METHYLTRANSFERASE 3B PLAYS A PROTECTIVE ROLE AGAINST HEPATOCARCINOGENESIS CAUSED BY CHRONIC INFLAMMATION VIA MAINTAINING MITOCHONDRIAL HOMEOSTASIS. MOST HEPATOCELLULAR CARCINOMAS (HCCS) DEVELOP ON THE BASIS OF CHRONIC HEPATITIS, BUT THE MECHANISM OF EPIGENETIC REGULATION IN INFLAMMATORY HEPATOCARCINOGENESIS HAS YET TO BE ELUCIDATED. AMONG DE NOVO DNA METHYLTRANSFERASES (DNMTS), DNMT3B HAS LATELY BEEN REPORTED TO ACT SPECIFICALLY ON ACTIVELY TRANSCRIBED GENES, SUGGESTING THE POSSIBILITY THAT IT PLAYS A ROLE IN THE PATHOGENESIS OF CANCER. WE CONFIRMED THAT DNMT3B ISOFORMS LACKING ITS CATALYTIC DOMAIN WERE HIGHLY EXPRESSED IN HCCS COMPARED WITH NON-TUMOROUS LIVER TISSUE. TO ELUCIDATE THE ROLE OF DNMT3B IN HEPATOCARCINOGENESIS, WE GENERATED A GENETICALLY ENGINEERED MOUSE MODEL WITH HEPATOCYTE-SPECIFIC DNMT3B DELETION. THE LIVER OF THE DNMT3B-DEFICIENT MICE EXHIBITED AN EXACERBATION OF THIOACETAMIDE-INDUCED HEPATITIS, PROGRESSION OF LIVER FIBROSIS AND A HIGHER INCIDENCE OF HCC COMPARED WITH THE LIVER OF THE CONTROL MICE. WHOLE-GENOME BISULFITE SEQUENCING VERIFIED A LOWER CG METHYLATION LEVEL IN THE DNMT3B-DEFICIENT LIVER, DEMONSTRATING DIFFERENTIALLY METHYLATED REGIONS THROUGHOUT THE GENOME. TRANSCRIPTOME ANALYSIS REVEALED DECREASED EXPRESSION OF GENES RELATED TO OXIDATIVE PHOSPHORYLATION IN THE DNMT3B-DEFICIENT LIVER. MOREOVER, PRIMARY HEPATOCYTES ISOLATED FROM THE DNMT3B-DEFICIENT MICE SHOWED REDUCED MITOCHONDRIAL RESPIRATORY CAPACITY, LEADING TO THE ENHANCEMENT OF OXIDATIVE STRESS IN THE LIVER TISSUE. OUR FINDINGS SUGGEST THE PROTECTIVE ROLE OF DNMT3B AGAINST CHRONIC INFLAMMATION AND HCC DEVELOPMENT VIA MAINTAINING MITOCHONDRIAL HOMEOSTASIS.	2020	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
14  332  41 ALTERATION OF EPIGENETIC PROFILE IN HUMAN HEPATOCELLULAR CARCINOMA AND ITS CLINICAL IMPLICATIONS. HEPATOCELLULAR CARCINOMA (HCC) IS A COMMON CANCER WORLDWIDE AND DEVELOPS AGAINST A BACKGROUND OF CHRONIC LIVER DAMAGE. A VARIETY OF HCC-RELATED GENES ARE KNOWN TO BE ALTERED BY GENETIC AND EPIGENETIC MECHANISMS. THEREFORE, INFORMATION REGARDING ALTERATION OF THE GENETIC AND EPIGENETIC PROFILES IN HCC IS ESSENTIAL FOR UNDERSTANDING THE BIOLOGY OF THIS TYPE OF TUMOR. METHYLATION AT CPG SITES IN GENE PROMOTERS IS KNOWN TO AFFECT THE TRANSCRIPTION OF THE CORRESPONDING GENES. ABNORMAL REGIONAL HYPERMETHYLATION IS OBSERVED IN THE 5' REGION OF SEVERAL TUMOR SUPPRESSOR GENES (TSGS) IN HCC, AND THIS HYPERMETHYLATION MAY PROMOTE CARCINOGENESIS THROUGH THE TRANSCRIPTIONAL INACTIVATION OF DOWNSTREAM TSGS. THE DNA DAMAGE INDUCED BY OXIDATION IS A TRIGGER OF ABNORMAL DNA METHYLATION AND INACTIVATION OF TSGS THROUGH RECRUITMENT OF THE POLYCOMB REPRESSIVE COMPLEX TO THE PROMOTER SEQUENCE. THUS, OXIDATIVE STRESS MAY BE RESPONSIBLE FOR THE EMERGENCE OF HCC FROM CHRONIC HEPATITIS AND LIVER CIRRHOSIS THROUGH THE EPIGENETIC ALTERATION OF TSGS. THERE HAVE BEEN SEVERAL ATTEMPTS TO APPLY EPIGENETIC INFORMATION TO THE DIAGNOSIS AND TREATMENT OF HCC. THE PREDICTIVE VALUE OF SELECTED METHYLATION EVENTS ON SURVIVAL IN HCC PATIENTS HAS BEEN REPORTED, AND THE METHYLATION PROFILE OF BACKGROUND LIVER COULD BE ASSOCIATED WITH RECURRENCE-FREE SURVIVAL OF HCC PATIENTS WHO HAVE UNDERGONE HEPATECTOMY. ANOTHER STUDY DETECTED METHYLATED DNA FROM HCC CELLS IN SERUM, AND THE CIRCULATING TUMOR DNA WAS REGARDED AS A POTENTIAL TUMOR MARKER. IN ADDITION, SEVERAL TRIALS OF HCC THERAPY HAVE TARGETED THE EPIGENETIC MACHINERY AND WERE BASED UPON COMPREHENSIVE ANALYSES OF DNA METHYLATION OF THIS TYPE OF TUMOR. HERE, WE PRESENT AN OVERVIEW OF RESEARCH REGARDING DNA METHYLATION STATUS IN HUMAN HCC AND DESCRIBE THE CLINICAL APPLICATION OF EPIGENETIC INFORMATION TO HCC.	2014	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           
15 4903  36 P16 PROMOTER HYPERMETHYLATION IN HUMAN HEPATOCELLULAR CARCINOMA WITH OR WITHOUT HEPATITIS VIRUS INFECTION. BACKGROUND: EPIGENETIC ALTERATION THROUGH METHYLATION IS ONE OF THE MOST IMPORTANT STEPS IN CARCINOGENESIS. HOWEVER, THE RELATION BETWEEN HEPATITIS VIRUS INFECTION AND EPIGENETIC ALTERATIONS IS POORLY UNDERSTOOD. METHODS: SIXTEEN PATIENTS WITHOUT HEPATITIS B VIRUS (HBV) AND HEPATITIS C VIRUS (HCV) AND 35 PATIENTS WITH HBV OR HCV WHO UNDERWENT LIVER RESECTION FOR HEPATOCELLULAR CARCINOMA (HCC) WERE STUDIED. MUTATION OF P53 WAS DETECTED BY DIRECT SEQUENCING. METHYLATION STATUS OF P16 WAS EVALUATED IN TUMOR AND NONCANCEROUS LIVER TISSUES BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. RESULTS: IN HCC WITHOUT HBV AND HCV, P53 MUTATIONS WERE DETECTED IN 5 (31%) OF 16 HCCS. METHYLATION OF P16 PROMOTER WAS DETECTED IN 2 (25%) OF 8 MODERATELY DIFFERENTIATED HCCS, 6 (75%) OF 8 POORLY DIFFERENTIATED HCCS, AND NONE OF 16 NONCANCEROUS TISSUE SPECIMENS. IN HCC WITH HBV OR HCV, P53 MUTATIONS WERE DETECTED IN 8 (23%) OF 35 HCCS. METHYLATION OF P16 PROMOTER WAS DETECTED IN 2 (100%) OF 2 WELL-DIFFERENTIATED HCCS, 13 (76%) OF 17 MODERATELY DIFFERENTIATED HCCS, 12 (75%) OF 16 POORLY DIFFERENTIATED HCCS, AND 9 (26%) OF 35 NONCANCEROUS LIVER TISSUE SPECIMENS. CONCLUSIONS: OUR RESULTS SUGGEST THAT HEPATITIS VIRUSES MIGHT INDUCE METHYLATION OF P16 PROMOTER IN LIVER WITH CHRONIC INFLAMMATION, BEFORE APPEARANCE OF HCC.	2004	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
16 6723  43 VITAMIN D RECEPTOR GENE METHYLATION IN HEPATOCELLULAR CARCINOMA. WORLDWIDE, HEPATOCELLULAR CARCINOMA (HCC) IS THE MAJOR SUBTYPE OF PRIMARY LIVER CANCERS. HCC IS TYPICALLY DIAGNOSED LATE IN ITS COURSE. WITH RESPECT TO CANCER, THE GENOMIC ACTIONS OF VITAMIN D ARE MEDIATED THROUGH BINDING TO THE VITAMIN D RECEPTOR (VDR), WHICH ALLOWS IT TO MODULATE THE EXPRESSION OF GENES IN A CELL-AND TISSUE-SPECIFIC MANNER. EPIGENETICS IS A RAPIDLY EVOLVING FIELD OF GENETIC STUDY APPLICABLE TO HCC. CHANGES IN DNA METHYLATION PATTERNS ARE THOUGHT TO BE EARLY EVENTS IN HEPATOCARCINOGENESIS. CURCUMIN HAS GREAT POTENTIAL AS AN EPIGENETIC AGENT. ACCORDINGLY, THE CURRENT STUDY HAS BEEN DESIGNED TO STUDY THE METHYLATION STATUS OF VDR GENE PROMOTER FOR THE FIRST TIME IN HCC AIMING TO FIND ITS CLINICAL SIGNIFICANCE AND POTENTIAL SCREENING ROLE IN CHRONIC LIVER DISEASE (CLD). ADDITIONALLY, WE AIMED TO INVESTIGATE, THE EFFECT OF CURCUMIN ON HCC CELL LINE, AIMING TO DISCOVER NEW THERAPEUTIC TARGETS THROUGH EPIGENETICS. THIS STUDY WAS CONDUCTED ON 45 FORMALIN-FIXED, PARAFFIN-EMBEDDED LIVER TISSUE BLOCKS INCLUDING 15 HCC SAMPLES (GROUP A), 15 CLD SAMPLES (GROUP B) AND 15 APPARENTLY NORMAL TISSUE TAKEN FROM AROUND BENIGN LESIONS (GROUP C). METHYLATION SPECIFIC RESTRICTION DIGESTION AND QPCR WERE DONE ON ALL SAMPLES AFTER DNA EXTRACTION. THE PERCENTAGE OF VDR GENE PROMOTER METHYLATION WAS SIGNIFICANTLY HIGHER IN THE HCC GROUP COMPARED TO BOTH CLD AND CONTROL GROUPS (P < 0.01). VDR PROMOTER METHYLATION BY (MS-QPCR) WAS DECREASED AND THE RELATIVE EXPRESSION OF VDR BY (QRT-PCR) WAS MARKEDLY INCREASED IN A DOSE-DEPENDENT FASHION IN CELLS GROWN IN CURCUMIN-ADEQUATE MEDIUM. IN CONCLUSION, THIS STUDY MAY OPEN A NEW GATE FOR THE USE OF VDR PROMOTER METHYLATION AS A POTENTIAL BIOMARKER IN HCC.	2018	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
17 4056  37 MAPPING THE HETEROGENEITY OF HISTONE MODIFICATIONS ON HEPATITIS B VIRUS DNA USING LIVER NEEDLE BIOPSIES OBTAINED FROM CHRONICALLY INFECTED PATIENTS. COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) FORMS THE BASIS FOR REPLICATION AND PERSISTENCE OF HEPATITIS B VIRUS (HBV) IN THE CHRONICALLY INFECTED LIVER. WE HAVE PREVIOUSLY SHOWN THAT VIRAL TRANSCRIPTION IS SUBJECT TO REGULATION BY POSTTRANSLATIONAL MODIFICATIONS (PTMS) OF HISTONE PROTEINS BOUND TO CCCDNA THROUGH ANALYSIS OF DE NOVO HBV-INFECTED CELL LINES. WE NOW REPORT THE SUCCESSFUL ADAPTATION OF THIS CHROMATIN IMMUNOPRECIPITATION SEQUENCING (CHIPSEQ) APPROACH FOR ANALYSIS OF FINE-NEEDLE PATIENT LIVER BIOPSY SPECIMENS TO INVESTIGATE THE ROLE OF HISTONE PTMS IN CHRONICALLY HBV-INFECTED PATIENTS. USING 18 SPECIMENS FROM PATIENTS IN DIFFERENT STAGES OF CHRONIC HBV INFECTION, OUR WORK SHOWS THAT THE PROFILE OF HISTONE PTMS IN CHRONIC INFECTION IS MORE NUANCED THAN PREVIOUSLY OBSERVED IN IN VITRO MODELS OF ACUTE INFECTION. IN LINE WITH OUR PREVIOUS FINDINGS, WE FIND THAT THE MAJORITY OF HBV-DERIVED SEQUENCES ARE ASSOCIATED WITH THE ACTIVATING HISTONE PTM H3K4ME3. HOWEVER, WE SHOW A STRIKING INTERPATIENT VARIABILITY OF ITS DEPOSITION IN THIS PATIENT COHORT CORRELATED WITH VIRAL TRANSCRIPTION AND PATIENT HBV EARLY ANTIGEN (HBEAG) STATUS. UNEXPECTEDLY, WE DETECTED DEPOSITION OF THE CLASSICAL INHIBITORY HISTONE PTM H3K9ME3 ON HBV-DNA IN AROUND HALF OF THE PATIENT BIOPSY SPECIMENS, WHICH COULD NOT BE LINKED TO REDUCED LEVELS OF VIRAL TRANSCRIPTS. OUR RESULTS SHOW THAT CURRENT IN VITRO MODELS ARE UNABLE TO FULLY RECAPITULATE THE COMPLEX EPIGENETIC LANDSCAPE OF CHRONIC HBV INFECTION OBSERVED IN VIVO AND DEMONSTRATE THAT FINE-NEEDLE LIVER BIOPSY SPECIMENS CAN PROVIDE SUFFICIENT MATERIAL TO FURTHER INVESTIGATE THE INTERACTION OF VIRAL AND HOST PROTEINS ON HBV-DNA.IMPORTANCE HEPATITIS B VIRUS (HBV) IS A MAJOR GLOBAL HEALTH CONCERN, CHRONICALLY INFECTING MILLIONS OF PATIENTS AND CONTRIBUTING TO A RISING BURDEN OF LIVER DISEASE. THE VIRAL GENOME FORMS THE BASIS FOR CHRONIC INFECTION AND HAS BEEN SHOWN TO BE SUBJECT TO REGULATION BY EPIGENETIC MECHANISMS, SUCH AS POSTTRANSLATIONAL MODIFICATION OF HISTONE PROTEINS. HERE, WE CONFIRM AND EXPAND ON PREVIOUS RESULTS BY ADAPTING A HIGH-RESOLUTION TECHNIQUE FOR ANALYSIS OF HISTONE MODIFICATIONS FOR USE WITH PATIENT-DERIVED FINE-NEEDLE LIVER BIOPSY SPECIMENS. OUR WORK HIGHLIGHTS THAT THE SITUATION IN VIVO IS MORE COMPLEX THAN PREDICTED BY CURRENT IN VITRO MODELS, FOR EXAMPLE, BY SUGGESTING A NOVEL, NONCANONICAL ROLE OF THE HISTONE MODIFICATION H3K9ME3 IN THE HBV LIFE CYCLE. IMPORTANTLY, ENABLING THE USE OF FINE-NEEDLE LIVER BIOPSY SPECIMENS FOR SUCH HIGH-RESOLUTION ANALYSES MAY FACILITATE FURTHER RESEARCH INTO THE EPIGENETIC REGULATION OF THE HBV GENOME.	2019	

18 4677  40 NEW LNCRNAS IN CHRONIC HEPATITIS C PROGRESSION: FROM FIBROSIS TO HEPATOCELLULAR CARCINOMA. HEPATOCELLULAR CARCINOMA (HCC) IS THE THIRD LEADING CAUSE OF CANCER-RELATED DEATH IN THE WORLD, AND ABOUT 80% OF THE CASES ARE ASSOCIATED WITH HEPATITIS B OR C. GENETIC AND EPIGENETIC ALTERATIONS ARE ACCUMULATED OVER DECADES OF CHRONIC INJURY AND MAY AFFECT THE FUNCTIONING OF TUMOR SUPPRESSOR GENES AND PROTOONCOGENES. STUDIES HAVE EVIDENCED THE ROLE OF LONG NON-CODING RNAS (LNCRNA) WITH ONCOGENIC OR TUMOR SUPPRESSOR ACTIVITIES, SUGGESTING A GREAT POTENTIAL IN THE TREATMENT, DIAGNOSIS OR INDICATOR OF PROGNOSIS IN CANCER. IN THIS CONTEXT, THE AIM OF THIS STUDY WAS TO EVALUATE THE GLOBAL EXPRESSION PROFILE LNCRNA IN HEPATIC TISSUE SAMPLES WITH DIFFERENT STAGES OF FIBROSIS ASSOCIATED WITH CHRONIC HEPATITIS C, HCC AND NORMAL LIVER, IN ORDER TO IDENTIFY NEW LNCRNAS THAT COULD CONTRIBUTE TO STUDY THE PROGRESSION OF HEPATIC FIBROSIS TO HCC ASSOCIATED WITH CHRONIC HEPATITIS C. RNA-SEQ WAS PERFORMED ON ILLUMINA NEXTSEQ PLATFORM TO IDENTIFY LNCRNAS EXPRESSED DIFFERENTLY IN 15 PATIENTS WITH CHRONIC HEPATITIS C, THREE PATIENTS WITH HCC AND THREE NORMAL LIVER SPECIMENS. WHEN THE PATHOLOGICAL TISSUES (FIBROSIS AND CARCINOMA) WERE COMPARED TO NORMAL HEPATIC TISSUE, WERE IDENTIFIED 2, 6 E 34 DIFFERENTIALLY EXPRESSED LNCRNAS IN MODERATE FIBROSIS, ADVANCED FIBROSIS AND HCC, RESPECTIVELY. THE CARCINOMA GROUP HAD THE HIGHEST PROPORTION OF DIFFERENTIALLY EXPRESSED LNCRNA (34) AND OF THESE, 29 WERE EXCLUSIVE IN THIS TYPE OF TISSUE. A HEAT MAP OF THE DEREGULATED LNCRNA REVEALED DIFFERENT EXPRESSION PATTERNS ALONG THE PROGRESSION OF FIBROSIS TO HCC. THE RESULTS SHOWED THE DEREGULATION OF SOME LNCRNA ALREADY CLASSIFIED AS TUMOR SUPPRESSORS IN HCC AND OTHER CANCERS, AS WELL AS SOME UNPUBLISHED LNCRNA WHOSE FUNCTION IS UNKNOWN. SOME OF THESE LNCRNAS ARE DYSREGULATED SINCE THE EARLY STAGES OF LIVER INJURY IN PATIENTS WITH HEPATITIS C, OTHERS OVEREXPRESSED ONLY IN TUMOR TISSUE, INDICATING THEMSELVES AS CANDIDATES OF MARKERS OF FIBROSIS PROGRESSION OR TUMOR, WITH POTENTIAL CLINICAL APPLICATIONS IN PROGNOSIS AS WELL AS A THERAPEUTIC TARGET. ALTHOUGH THERE ARE ALREADY STUDIES ON LNCRNA IN HEPATOCELLULAR CARCINOMA, THIS IS THE FIRST STUDY CONDUCTED IN SAMPLES EXCLUSIVELY OF HCV-RELATED LIVER AND HCV HCC.	2020	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
19  153  43 ABERRANT METHYLATION OF MULTIPLE TUMOR SUPPRESSOR GENES IN AGING LIVER, CHRONIC HEPATITIS, AND HEPATOCELLULAR CARCINOMA. ABERRANT DNA METHYLATION IS AN IMPORTANT EPIGENETIC ALTERATION IN HEPATOCELLULAR CARCINOMA (HCC). HOWEVER, THE MOLECULAR PROCESSES UNDERLYING THE METHYLATOR PHENOTYPE AND THE CONTRIBUTION OF HEPATITIS VIRUSES ARE POORLY UNDERSTOOD. THE CURRENT STUDY IS A COMPREHENSIVE METHYLATION ANALYSIS OF HUMAN LIVER TISSUE SPECIMENS. A TOTAL OF 176 LIVER TISSUES, INCLUDING 77 PAIRS OF HCCS AND MATCHING NONCANCEROUS LIVER AND 22 NORMAL LIVERS, WERE ANALYZED FOR METHYLATION. METHYLATION OF 19 EPIGENETIC MARKERS WAS QUANTIFIED, AND THE RESULTS WERE CORRELATED WITH DIFFERENT DISEASE STATES AND THE PRESENCE OR ABSENCE OF HEPATITIS B VIRUS (HBV) AND HEPATITIS C VIRUS (HCV) INFECTIONS. BASED ON METHYLATION PROFILES, THE 19 LOCI WERE CATEGORIZED INTO 3 GROUPS. NORMAL LIVER TISSUES SHOWED METHYLATION PRIMARILY IN GROUP 1 LOCI (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, P16, APC), WHICH WAS SIGNIFICANTLY HIGHER THAN GROUP 2 (CDH1, RUNX3, RIZ1, SFRP2, MINT31) AND GROUP 3 MARKERS (COX2, MINT1, CACNA1G, RASSF2, MINT2, REPRIMO, DCC) (P < 0.0001). NONCANCEROUS LIVERS DEMONSTRATED INCREASED METHYLATION IN BOTH GROUP 1 AND GROUP 2 LOCI. METHYLATION WAS SIGNIFICANTLY MORE ABUNDANT IN HCV-POSITIVE LIVERS COMPARED WITH NORMAL LIVER TISSUES. CONVERSELY, HCC SHOWED FREQUENT METHYLATION AT EACH LOCUS INVESTIGATED IN ALL 3 GROUPS. HOWEVER, THE GROUP 3 LOCI SHOWED MORE DENSE AND FREQUENT METHYLATION IN HCV-POSITIVE CANCERS COMPARED WITH BOTH HBV-POSITIVE CANCERS AND VIRUS-NEGATIVE CANCERS (P < 0.0001). CONCLUSION: METHYLATION IN HCC IS FREQUENT BUT OCCURS IN A GENE-SPECIFIC AND DISEASE-SPECIFIC MANNER. METHYLATION PROFILING ALLOWED US TO DETERMINE THAT ABERRANT METHYLATION IS COMMONLY PRESENT IN NORMAL AGING LIVERS, AND SEQUENTIALLY PROGRESSES WITH ADVANCING STAGES OF CHRONIC VIRAL INFECTION. FINALLY, OUR DATA PROVIDE EVIDENCE THAT HCV INFECTION MAY ACCELERATE THE METHYLATION PROCESS AND SUGGESTS A CONTINUUM OF INCREASING METHYLATION WITH PERSISTENT VIRAL INFECTION AND CARCINOGENESIS IN THE LIVER.	2008	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
20 5357  40 REACTIVE OXYGEN SPECIES INDUCE EPIGENETIC INSTABILITY THROUGH THE FORMATION OF 8-HYDROXYDEOXYGUANOSINE IN HUMAN HEPATOCARCINOGENESIS. CHRONIC HEPATITIS C (CHC) TRIGGERS OXIDATIVE STRESS AND CONTRIBUTES TO THE EMERGENCE OF HEPATOCELLULAR CARCINOMA (HCC). WE PREVIOUSLY REPORTED THAT TUMOR SUPPRESSOR GENE (TSG) METHYLATION IS A CRITICAL FACTOR DURING THE EARLY STAGES OF HEPATOCARCINOGENESIS. IN THIS STUDY, WE CLARIFY THE ASSOCIATION BETWEEN OXIDATIVE STRESS AND EPIGENETIC ALTERATIONS DURING HEPATOCARCINOGENESIS. WE EXAMINED DNA OXIDATION AND METHYLATION PROFILES IN 128 LIVER BIOPSY SAMPLES FROM CHC PATIENTS. THE DNA OXIDATION AND METHYLATED TSG NUMBERS WERE QUANTIFIED USING IMMUNOHISTOCHEMICAL ANALYSIS OF 8-HYDROXYDEOXYGUANOSINE (8-OHDG) AND QUANTITATIVE PCR FOR 11 TSGS, RESPECTIVELY. THE QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION-PCR (CHIP-QPCR) ASSAY IN HEPG2 AND FETAL LIVER HC CELLS TREATED WITH H2O2 WAS USED TO QUANTIFY TRIMETHYL-H3K4, ACETYLATED-H4K16 (AN ACTIVE CHROMATIN MARKER), TRIMETHYL-H3K27 (A REPRESSIVE CHROMATIN MARKER) AND 8-OHDG. WE ANALYZED 30 PROMOTERS OF 25 DIFFERENT TSGS BY QPCR. THE HIGH LEVELS OF 8-OHDG WAS THE ONLY VARIABLE THAT WAS SIGNIFICANTLY ASSOCIATED WITH THE INCREASED NUMBER OF METHYLATED TSGS IN CHC (P < 0.0001). THE CHIP-QPCR REVEALED THAT AFTER H2O2 TREATMENT OF THE CELL LINES, THE 8-OHDG-BOUND PROMOTERS SHOWED A MODIFICATION FROM AN ACTIVE CHROMATIN (TRIMETHYL-H3K4 AND ACETYLATED-H4K16 DOMINANT) TO A REPRESSIVE CHROMATIN (TRIMETHYL-H3K27 DOMINANT) STATUS. WE CONCLUDE THAT OXIDATIVE STRESS ALTERS THE CHROMATIN STATUS, WHICH LEADS TO ABNORMAL METHYLATION OF TSGS, AND CONTRIBUTES TO HEPATOCARCINOGENESIS IN CHC PATIENTS.	2013