| Part ⅠArray-based Comparative Genomic Hybridization and Isobaric Tagging Reagent (iTRAQ) Quantitative Proteomics Analysis of Hepatocellular Carcinoma Exposed to Hepatitis B Virus and Aflatoxin B1in Guangxi AreaObjective This study was trying to find the specific molecular biological markers in the development of HBV-related and AFB1-related hepatocellular carcinoma, and to explore the molecular mechanism of the synergetic hepatocarcinogenesis effect of both HBV and AFB1by studying the difference of whole-genome profiles and protein expression profiles in hepatocellular carcinoma which was exposed to hepatitis B virus and Aflatoxin B1in Guangxi area.Methods First of all,32cases from different districts of Guangxi province who were consequentially hospitalized for radical resection of HCC in the department of hepatobiliary surgery of the first affiliated hospital to Guangxi medical university from February2008to September2010were labeled as four categories based on their biomarkers of HBV and AFB1exposure:group A (HBV(+)/AFB1(+)), group B (HB V(+)/AFB1(-)), group C (HBV(-)/AFB1(+)) and group D (HBV(-)/AFB1(-)). In our study, we defined the criterion of HBV(+) as the patient who were HBsAg-positive, and defined the criterion of AFB1(+) as the patient who had a specific AGGArg'AGTSer (or AGGArg'AGCSer)mutation in Codon249of the p53gene in tumor tissue with/without the immunohistochemically positive AFB1-DNA adduct. And normal hepatic tissue from10cases of hepatic hemangioma, liver resection and liver transplant donor were chosen as normal control group. Array-based comparative genomic hybridization (aCGH) and Isobaric Tagging Reagent (iTRAQ) Quantitative Proteomics combined with2DLC-MS/MS were used to analyse the chromosomal copy number changes and differentially expressed proteins of the tumor tissues. And then the results of Array CGH and proteomics were validated by Reverse Transcriptase PCR and Western-blot.Results (1) A total of573CNAs (184gains and389losses) were found in32HCC samples in all. Common alterations included gains in1q,4p,5p,6p,7p,8q,10p,17q,20p,20q and X, and losses in1p,2q,4q,8p,9p,10q,11q,13q,14q,16p,16q,17p,19p,19q,21q,22q and Y repetitively.(2) There were some recurrently altered regions (RARs) in32HCC samples. In total,25RARs (including8RAR gains and17RAR losses) were detected in our study.8gain RARs were1q21.1-q44,5p13.2-p15.3,6p12.1-p25.2,7q21.1-q35,8q11.2-q24.3,17q12-q25.2,18q12.3-q22.3and X.17loss RARs were1p31.1-p36.2, 2q23.2-q37.2,4q12-q35.2,6q14.1-q26,8p12-p23.2,9p21.1-p24.2,10q21.3-q26.2,13ql2.1-q21.1,14q21.3-q32.2,16p12.1-p13.2,16ql2.1-q24.1,17p12-p13.3,19p13.1-p13.3,19q13.2-q13.4,21q21.3-q22.2,22q11.2-q13.2and Y, respectively. In these25RARs, we found that4RARs (2q23.2-q37.2,18q12.3-q22.3,19p13.1-p13.3and Y) had rarely been identified in other studies. The incidence of losses in8p12-p23.2was significantly higher in high tumor grade(Ⅲ-Ⅳ) and high tumor stage (Ⅲ-Ⅳ) than low tumor grade(Ⅰ-Ⅱ) and low tumor stage(Ⅰ-Ⅱ)(p<0.05). In addition, the incidence of gains in1q21.1-q44was significantly higher in low tumor stage(Ⅰ Ⅱ)than high tumor grade stage (Ⅲ-Ⅳ)(p<0.05).(3) The incidence of losses in RARs4q13.3-q35.2and13q12.1-q21.2and gain in RARs7q21.1-q35were very high in groupA (100%,70%,70%respectively), but very low in groupD (only33.3%,0,16.7%repetitively).(4) Cox regression model showed that the serum AFP level, liver cirrhosis, invasion and metastasis, TNM tumor stage, as well as loss of8p12-p23.2and6p12.1-p25.2were significantly and independently unfavorable prognostic factors (all p<0.05).(5) In total,22high-level copy number changes (HLCNCs) including14amplifications and9homozygous deletions (HD) were identified in32HCC cases.The amplification on1q21.1-q24.2,1q31.1-q44and8q11.2-q24.3were all observed in5cases, and the homozygous deletions on4q22-q24、13q14.2and Y were detected in5cases,4cases and6cases repetitively.(6) In iTRAQ combined with2DLC-MS/MS quantitative proteomics analysis, a total of117unique differentially expressed proteins including53up-regulated proteins and64up-regulated proteins were identified in four groups (106,97,104and74proteins in group A, B, C and D respectively). The117differentially expressed proteins were involved in14kinds of biological process which included detoxification and drug metabolism pathway, anti-apoptosis pathway, antigen processing and presentation pathway, glycolysis pathway, etc.When comparing the differentially expressed proteins profiles with the corresponding genomic DNA copy number changes, we found that47of all117differentially expressed proteins mapped within recurrently altered regions (RARs).(7) Among117differentially expressed proteins, we found that9of them were heat shock proteins or chaperones (HSP90AA1, HSP90AB1, GRP78, HSPA1A, GRP75, HSPA8, HSPD1, HSPE1and HSPB1). They were up-regulated in group A, B and C(some of them also displayed up-regulated in group D), and most of them were involved in anti-apoptosis pathway and antigen processing and presentation pathways. Besides,15proteins (GSTA1, CYP2A13, ADH4, ADH1C, HADH, CYP2E1, CYP27A1, ADH1A, ADH6, EPHX2, EPHX1, AKR1C1, AKR1C4, AKR1B10, AOX1) were involved in the detoxification and drug metabolism pathway. Except for AKR1C1, AKR1C4and AKRIB10, all of the other12proteins were down-regulated in the group A, and more than half of them also displayed down-regulated in group B and C. Interestingly,11proteins mentioned above mapped within RARs. They were CYP2A13, ADH4, ADH1C, HADH, CYP2E1, CYP27A1, ADH1A, ADH6, EPHX2, AKRIBIO and AOX.(8) The Reverse Transcriptase PCR result showed that the mean expression level of AKR1B10mRNA in group A(1.39±0.69) was significantly higher than group B(0.88±0.28), C(1.03±0.42) and D (0.58±0.14)(all p<0.05). The group C also showed significantly higher expression level of AKR1B10mRNA than group D(p=0.011). The Western-blot results showed that the mean expression level of AKRIB10protein in group A (2.79±1.30) was significantly higher than group B(2.03±0.98) and D(1.65±0.49)(all p<0.05) but not than group C (2.23±1.16, p>0.05).Conclusions1. The chromosomal alterations of HCC cases had the multiplicity in Guangxi area. The loss of19p13.1-p13.3may be one of unique genetics characteristics for HCC in Guangxi area.2. The gains of1q21.1-q44may be an early event of hepatocarcinogenesis, whereas the loss of8p12-p23.2may occur as a later event in HCC progression and relate to the pathological grade and bad prognosis. The loss of4q12-q35.2 and13q12.1-q21.land the gain of7q21.1-q35may be associated with the synergetic hepatocarcinogenesis effect of both HBV and AFB1.3. There were some general characteristics and varieties in protein expression among these four subgroups:HB V(+)/AFB1(+), HBV(+)/AFB1(-), HBV(-)/AFB1(+) and HBV(-)/AFB1(-).117differential proteins involved in detoxification and drug metabolism, anti-apoptosis, antigen processing and presentation pathway and glycolysis pathway, etc. It implied that the occurrence and development of HCC was a complicated process which polygene and poly pathway involved in.4. The up-regulated expression of heat shock protein and the down-regulated expression of most protein enzymes related to detoxification and drug metabolism were the common molecular biological events of HCC associate with the exposure to HBV and AFB1. And this indicated that the synergetic hepatocarcinogenesis effect of both HBV and AFB1related to gene dysregulation of heat shock protein and protein enzymes related to detoxification and drug metabolism.5. The overexpression of AKR1B10was found in HBV/AFB1-related and AFB1-related HCC cases. This implied that the overexpression of AKR1B10may involve in the hepatocarcinogenesis process and be one of the molecular biological markers associated with HCC cases exposed to AFB1which has the potentially clinical therapy value. Part IIThe Preliminary Analysis of Mutation and Expression of Gene P53, CTNNB1and PTEN among cases with Hepatocellular Carcinoma Exposed to Hepatitis B Virus and Aflatoxin B1in Guangxi AreaObjective This study was to investigate the mutation and expression of P53, CTNNB1, PTEN gene among cases with hepatocellular carcinoma exposed to hepatitis B Virus and aflatoxin B1in Guangxi area, and to explore whether these cases had the molecular biological characteristics associated with the exposure to hepatitis B virus and aflatoxin B1, and to discuss the relationship between the exposure to hepatitis B Virus and aflatoxin Bland the occurrence and development of hepatocellular carcinoma.Methods108cases from different districts of Guangxi province who were consequentially hospitalized for radical resection of HCC in the department of hepatobiliary surgery of the first affiliated hospital ofGuangxi medical university from April2008to June2010were labeled as four groups based on their biomarkers of HBV and AFB1exposure:group A(HBV(+)/AFB1-DNA(+)), group B(HBV(+)/AFB1-DNA(-)), group C (HBV(-)/AFB1-DNA(+)) and group D (HBV(-)/AFB1-DNA(-)). In our study, we defined the criterion of HBV (+) as the patient who was HBsAg-positive, and defined the criterion of AFB1(+) as the patient who had immunohistochemically positive AFB1-DNA adduct in tumor tissue. And normal hepatic tissue from20cases of hepatic hemangioma, liver resection and liver transplant donor were chosen as normal control group. And the mutation was detected for the entire sequence of P53gene, Exon3of (3-catenin gene and Exon4,5and8of PTEN by PCR-DNA sequencing. The expression of gene P53, β-catenin and PTEN were detected by RT-PCR and/or immunohistochemistry.Results (1) The DNA sequencing results of P53gene were as followed. Among108HCC cases, the mutation of P53gene was found in65cases (60.2%). The mutation rate of Codon249in Exon7was highest (41.6%). Besides, Codon236changed from TAC to TGC in one case and Code261changed from AGT to AAT in P53gene in two cases. The mutations of Exon5in P53gene were as followed:one case with Codon157GTC'CTC, one case with Codon177CCC'CCG, six cases with Codon179CAT'CGT or CTT and four cases with Codon205TAT'TGT. The mutations of Exon5in P53gene were as followed:one case with Codon214CAT'CGT and two cases with Codon219ATG'GTG. Codon281in Exon8changed from GAC to GAG in one case and Codon306in Exon8changed from CGA to CGT in one case. One case was found the mutation (AAG'AAA) in Codon320of Exon9. Two cases were found the mutation (AGG'AGT) in Codon249of Exon7in108adjacent normal tissue pairs and20normal cases. The mutation rate of P53gene in group A and C were respectively87.5%and68.4%, which were significantly higher than in group B and D(all p<0.05).(2) The DNA sequencing results of Exon3in β-catenin gene were as followed.12out of108cases (11.15) were found mutation. One case was found the mutation in Coden32(GAC'GGC). Four cases were found the mutation (TCT'TAT) and one case was found the mutation (TCT'CCT) and two cases were found the mutation (TCT'GCT) in Coden37. Coden41changed from ACC to GCC in4cases. The mutation rates of Exon3in β-catenin gene were14.5%,3.7%,21.1%and0in group A, B, C and D, respectively. There was no significant difference in the mutation rate of Exon3in β-catenin gene among these four group(p>0.05).(3) No mutation was found in Exon4,5and8in PTEN gene. But61out of108cases (56.4%) were found the loss in splicing sites between Exon4and Intron4. There was no significant difference in loss rate among group A, group B, group C and group D, which were60.4%,62.9%,47.3%and46.6%, respectively.(4) The positive expression rates of P53protein in108HCC cases and20normal cases were76.8%(83/108) and10%(2/10) respectively, which had significant difference (p<0.001). The positive expression rates of P53protein in group A, B, C and D were87.5%(42/48),66.7%(18/27),78.9%(15/19) and42.9%(8/14), respectively. There were significant differences in the positive expression rates of P53protein between group A and B (p=0.039), A and D (p=0.020) respectively. And there was no significant difference among other groups(p>0.05).(5) The immunohistochemical positive expression of β-catenin protein located at the cell membrane, cytoplasm and nucleus. There were significant differences in the positive expression rate of β-catenin protein between108HCC cases (70.0%) and20normal cases(15%)(p<0.001). Among75cases with the immunohistochemical positive expression of P-catenin protein,11cases (14.6%) were located at cell nucleus,43cases (57.3%) located at cell cytoplasm, and21cases (28.0%) located at cell membrane. The immunohistochemical positive expression of β-catenin protein was located at cell membrane in3normal cases. All12cases with β-catenin gene mutation were found the immunohistochemical positive expression of β-catenin protein:8cases at cell cytoplasm,1case at cell nucleus and3cases at cell membrane. The positive expression rates of β-catenin protein in group A, B, C and D were70.8%(34/48),66.6%(18/27),78.9%(15/19) and57.1%(8/14) respectively, which had no significant difference among these4groups. The mean expression level of β-catenin gene mRNA in group A, B, C, D and control group were1.13±0.14,1.06±0.12,1.16±0.18,1.01±0.13and0.085±0.13respectively. There were significant differences between group A and C (p=0.028), A and D.(P=0.032). And there were significant differences between these four group and control group(all p<0.05).The mean expression level of PTEN gene mRNA in four subgroup A, B, C, D and control group were0.54±0.13,0.59±0.16,0.97±0.16,0.92±0.13and1.10±0.16respectively. There were significant differences between group A and D (p=0.028), C and D.(p=0.032). And there were significant differences between group A and C(p=0.002), A and D(p=0.032), B and C (p<0.001) and B and D(P=0.011). And there were significant differences between subgroup A, B and D and control group (all p<0.05).Conclusions (1) The HCC cases in Guangxi area had a variety of P53gene mutation and its mutation hotspot was located at Coden249in Exon7, which was one of the common molecular biological characteristics in HCC cases in Guangxi area.(2)The mutation of gene P53and Exon3in gene β-catenin may be one of the causes of its own gene overexpression. And the loss of gene PTEN may results in the down-regulated expression level of this gene.(3) The overexpression of gene P53and β-catenin of HCC cases may be associated with the exposure to aflatoxin B1in Guangxi area while the loss of gene PTEN may relate to the exposure to hepatitis B Virus. |
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