Association Of SNPs In DNA Repair Gene With HCC Susceptibility And Influence Of MIF Promoter Polymorphism On Prognosis Of HCC

Dominated by hepatocellular carcinoma (HCC), primary hepatocellular carcinoma(PHC) is one of the most common malignant tumors seriously threatening people’s health.The fatality rate of HCC has ranked the second and first place in urban and rural cancermortality respectively. Currently, some preliminary understandings to the possible causeshave been obtained, but its pathogenesis is not entirely clear. HBV and HCV infections arethe most important causes of HCC, and drinking, smoking and family history are allconsidered as the important dangerous factors for the occurrence of HCC.DNA repair damage gene plays an important role in maintaining the stability ofgenetic information. The hereditary defect of DNA repair access may damage the integrityof genes, transforming cells to tumor cells. DNA damage repair gene mononucleotidepolymorphism may change the structure and function of repair enzyme, and then affect thestability of genes and the occurrence of tumor.XRCC1and XPD genes are repair damage genes; XRCC1is mainly engaged in genebase excision repair, while XPD gene is mainly engaged in gene nucleotide excision repair.The mutability of single base of the locus leads to the change of expression protein infunction, which causes the occurrence of tumor. The study analyzes the polymorphism threelocus genes of XRCC1-194, XRCC1-280and XPD-312, expecting to disclose itsassociation with hepatocellular carcinoma susceptibility.So far, the diagnosis system on HCC which combines laboratory examination withalpha fetoprotein as an important marker and image technology has been developed to amature level, with a coincidence rate of pre-operative diagnosis and pathologic diagnosis of97.4%. Currently, hepatocellular carcinoma prognosis is mainly decided based on indicatorsof post-operative pathological type, transfer and recurrence, etc. And such decision can onlybe carried out after the tissue specimen is obtained after surgical operation. Differenthepatocellular carcinoma patients have different prognosis indicators. And causes for such difference are poorly understood now. Macrophage migration inhibition factor (MIF) isrelated to many tumors. Some even believe that it can be used as one important marker forrelevant tumor decision prognosis. Research indicates MIP promoter gene polymorphismmay affect the expression of MIP and the prognosis of tumor. No literature has reportedwhether MIF promoter gene polymorphism is related to the prognosis of hepatocellularcarcinoma and what the association is.Objective:1. Discuss the association of DNA repair gene XRCC1and XPD gene polymorphismwith HCC susceptibility.2. With the influence of gene polymorphism on the occurrence of hepatocellularcarcinoma under the condition of gender, age, HBV infection, smoking, drinking and familyhistory as the dangerous factors for disease occurrence, further state the relationshipbetween three locus polymorphism of XRCC1-194, XRCC1-280and XPD-312and thehepatocellular carcinoma risk of Chinese Han group.3. Analyze the association of CATT microsatellite polymorphism of MIF-794promotergene of HCC patients with relevant indicators of hepatocellular carcinoma prognosis,evaluate its value of prognosis decision after hepatectomy, and expect to provide possibilityfor HCC prognosis decision.Research contents and methods:1. Study on the association of XRCC1and XPD gene mononucleotide polymorphismwith hepatocellular carcinoma susceptibility.Adopt the hospital-based case-control research method. The research object is a groupof Han people from the same region. Members of the Case Group are HCC patientsdiagnosed by histopathology who received treatment in Daping Hospital from January2009to December2010. Members of the Control Group are individuals from the same regiontaking physical examinations in Daping Hospital in the same period and selected randomly.1.1Collect the peripheral venous blood of3-5ml of the research objects in theanti-freezing blood vessel with ACD (citric acid, sodium citrate and dextrose), and storethem in refrigerator at the temperature of－20℃after being numbered. Adopt the bloodgenome DNA extraction kit of Tiangen Bitotech Co., Ltd. and extract blood specimengenome DNA according to the product operating manual. 1.2Apply polymerase chain reaction-restricted fragment length polymorphisms(PCR-RFLP) technology and gene sequencing method to genotype decision.1.3Check and analyze with χ2the distribution difference of genotype of XRCC1Arg194Trp, XRCC1Arg280His and XPD Asp312Asn locus and distribution frequency ofallele in the Case Group and Control Group. Carry out comparative analysis with superiorallele (homozygote of wild type) as the comparison, and with unconditional logisticregression analysis method, calculate OR and the connection between its95%CI indicatingsingle genotype and the risk HCC occurrence and the connection between it and the risk ofHCC occurrence under the joint action of XRCC1and XPD genes.2. Influence of MIF promoter gene polymorphism on the prognosis of HCCThe HCC patient applied to MIF promoter analysis are HCC patients diagnosed byhistopathology receiving surgical operations in the Department Of Hepatobiliary Surgery ofDaping Hospital from January2009to June2011. No tumor metastasis has been observedin all research objects. Phone return visits were paid to the research objects after operations,and the observing time lasted for6to36months. B Ultrasound or CT reexamination wascarried out every3months to observe whether there is reoccurrence or transfer of tumor.Experimental observation indicators include TNM staging of patient tumor, tumorpathologic inspection for histological classification, survival time in observing time andtime of reoccurrence and transfer of tumor (including swelling and transfer of lymph node).2.1Collect the peripheral venous blood of3-5ml of the research object in theanti-freezing blood vessel with ACD (citric acid, sodium citrate and dextrose), and storethem in refrigerator at the temperature of－20℃after being numbered. Adopt the bloodgenome DNA extraction kit of Tiangen Bitotech Co., Ltd. and extract blood specimengenome DNA according to the product operating manual.2.2Apply bidirectional gene sequencing method to check the repeated sequence of-794CATT microsatellite of MIF gene promoter for genotype identification.3.3Check, analyze and compare with χ2the tumor prognosis indicators (tumordifferentiation degree, TNM staging, survival rate, reoccurrence and transfer) between5/5+5/6+6/6and7/X+8/X genotypes, and check and compare with T the difference ofaverage survival time between two genotypes.All the above statistical tests are bilateral probability test, all data processing adopts SPSS13.0statistical software for analysis, and P＜0.05is marked difference.Results:1. Study on the association of XRCC1and XPD gene mononucleotide polymorphismwith hepatocellular carcinoma (HCC) susceptibility1.1XRCC1194HCC group contains119cases of wild genotype (CC),115cases ofheterozygosis genotype (CT) and18cases of TT genotype. XRCC1194healthy ControlGroup contains129cases of wild genotype (CC),102cases of heterozygosis genotype (CT)and21cases of (TT) genotype. The results of unconditioned logistic regression analysis are:OR﹦1.17,95%CI﹦(0.87-1.59), P﹦0.42, which indicate that the risk of XRCC1194locus heterozygosis allelic genotype to be attacked by liver cancer is17%higher than wildhomozygosis allelic genotype, but there is no statistic otherness.1.2XRCC1280HCCcontains193cases of wild genotype (GG),53cases of heterozygosis genotype (GA) and6cases of AA genotype. XRCC1280healthy Control Group contains207cases of wildgenotype (GG),40cases of heterozygosis genotype (GA) and5cases of (AA) genotype.The results of unconditioned logistic regression analysis are: OR﹦1.41,95%CI﹦(0.96-2.06), P﹦0.15, which indicate that the risk of XRCC1280locus heterozygosis allelicgenotype to be attacked by liver cancer is41%higher than wild homozygosis allelicgenotype, but there is no statistic otherness.1.3XPD312liver cancer group contains192cases of wild genotype (GG),58cases of heterozygosis genotype (GA) and2cases ofAA genotype. XPD312healthy Control Group contains209cases of wild genotype (GG),42cases of heterozygosis genotype (GA) and1case of (AA) genotype. The results ofunconditioned logistic regression analysis are: OR﹦1.56,95%CI﹦(1.51-2.37), P﹦0.08,which indicate that the risk of XPD312locus heterozygosis allelic genotype to be attackedby liver cancer is1.56times of wild homozygosis allelic genotype, but there is no statisticotherness.1.4The results of stratified analysis taking age (Equal to or above50and below50)and sex as risk factors indicate that the3polymorphic loci of the two genes have nointeraction with sex and age and value P is bigger than0.05for both of them.1.5The results of stratified analysis taking drinking, smoking, surface antigen carryingand family history as risk factors indicate that there is no obvious difference of XRCC1-194Arg194Trp locus genotype occurrence frequency in the Case Group and Control Group. But for XRCC1-194Trp genotype exposing to the condition of family genetic factor, the resultsof unconditioned logistic regression analysis are: OR﹦2.21,95%CI﹦(0.97-5.02), P﹦0.15which indicate that the risk of HCC occurrence is1.2times of the healthy people. SinceP>0.05, there is no statistic otherness.1.6The results of stratified analysis taking drinking, smoking, surface antigen carryingand family history as risk factors indicate that XRCC1-280His increases the occurrencefrequency of HCC, and the results of unconditioned logistic regression analysis are: OR﹦1.51,95%CI﹦(0.99-2.30), P﹦0.10; OR﹦1.38,95%CI﹦(0.88-2.18), P﹦0.15; OR﹦1.68,95%CI﹦(1.08-2.60), P﹦0.04and OR﹦4.20，95%CI﹦(1.34-13.20), P﹦0.03. Althoughduring the exposure to drinking and smoking, XRCC1-280His increases the occurrencefrequency of HCC, the results have no statistic meaning for P>0.05. For people with HBVor the family history liver cancer, the occurrence frequency HCC is0.68times and3.2times of the healthy and P is smaller than0.05in both conditions.1.7The results of stratified analysis taking drinking, smoking, surface antigen carryingand family history as risk factors indicate that XRCC1-280His increases the occurrencefrequency of HCC, and the results of unconditioned logistic regression analysis are: OR﹦1.67,95%CI﹦(1.10-2.60), P﹦0.03OR﹦1.87,95%CI﹦(1.18-2.96), P﹦0.02; OR﹦1.96,95%CI﹦(1.24-3.10), P﹦0.01and OR﹦3.40,95%CI﹦(1.32-8.76), P﹦0.03。Theseresults indicate that drinking, smoking, HBV infection factor and family history of livercancer have interaction with XPD-312Asn, and they increase the attacking risk of livercancer, but since P lower than0.05in each condition, there is no statistic meaning.1.8Allelic genotype containing XRCC1194Trp＋XRCC1280His, XRCC1194Trp＋XPD312Asn and XRCC1280His＋XPD312Asn increase occurrence frequency of HCCand the results of unconditioned Logistic regression are: OR﹦1.72,95%CI﹦(1.01-2.95),P﹦0.10; OR﹦2.00,95%CI﹦(1.14-3.38), P﹦0.04and OR﹦3.38,95%CI﹦(1.64-6.94),P﹦0.00.2. Influence of MIF promoter gene polymorphism on the prognosis of HCC2.1This Case Group contains182male cases and59female cases aging from21to73,with an average age of50.1±9.3. It includes9microsatellite CATT genotypes withrepetitive sequence of5/5,5/6,5/7,5/8,6/6,6/7,6/8,7/7and7/8. The study results aredescribed as follows: There are19cases of5/5repetitive sequence case and the average survival time is20.61±7.97months. The Phase I, II, III, and IV respectively are6,6,4and3cases. Thehigh, intermediate, and low differentiation of tissue samples respectively are4,6and9cases. The transfer-recurrence is found on7cases and5cases die. There are32cases of5/6repetitive sequence case and the average survival time is19.97±8.09months. The Phase I,II, III, and IV respectively are12,7,7and6cases. The high, intermediate, and lowdifferentiations of tissue sample respectively are4,15and13cases. The transfer-recurrenceis found on15cases and9cases die. There are25cases of5/7repetitive sequence case andthe average survival time is16.16±7.36months. The Phase I, II, III, and IV respectively are4,7,8and6cases. The high, intermediate, and low differentiations of tissue samplerespectively are2,6and17cases. The transfer-recurrence is found on14cases and10cases die. There are4cases of5/8repetitive sequence case and the average survival time is19.25±8.77months. The Phase I, II, III, and IV respectively are0,1,0and3cases. Thehigh, intermediate, and low differentiations of tissue sample respectively are1,0and3cases. The transfer-recurrence is found on3cases and2cases die.There are51cases of6/6repetitive sequence case and the average survival time is18.86±7.40months. The Phase I, II, III, and IV respectively are18,12,13and8cases. Thehigh, intermediate, and low differentiations of tissue sample respectively are4,21and26cases. The transfer-recurrence is found on20cases and11cases die. There are20cases of6/7repetitive sequence case and the average survival time is15.36±6.28months. The PhaseI, II, III, and IV respectively are14,12,16and16cases. The high, intermediate, and lowdifferentiations of tissue sample respectively are2,8and48cases. The transfer-recurrenceis found on37cases and25cases die. There is1case of6/8repetitive sequence case andthe average survival time is21months. The Phase I, II, III, and IV respectively are0,0,1and0case. The high, intermediate, and low differentiations of tissue sample respectivelyare0,0and1case. The transfer-recurrence is found on1case and1case die.There are25cases of7/7repetitive sequence case and the average survival time is15.80±7.18months. The Phase I, II, III, and IV respectively are8,3,7and7cases. Thehigh, intermediate, and low differentiations of tissue sample respectively are2,4and19cases. The transfer-recurrence is found on15cases and11cases die. There are26cases of7/8repetitive sequence case and the average survival time is14.85±6.69months. The Phase I, Ii, III and IV respectively are6,4,7and9cases. The high, intermediate, and lowdifferentiations of tissue sample respectively are2,5and19cases. The transfer-recurrenceis found on16cases and11cases die.2.2Comparative analysis is carried out on MIF-794CATT by grouping it to high andlow repetitive sequence (7/X+8/X,5/5+5/6+6/6). There is otherness in the5indexes ofdifferentiation, TNM phase, survival rate, reoccurrence and transfer and average survivaltime and P is smaller than0.05. The result is as follows:For (7/X+8/X) repetitive sequence case, its high-intermediate differentiation and lowdifferentiation respectively are32and107, while for (5/5+5/6+6/6), they respectively are54and48, and P=0.000. For (7/X+8/X) repetitive sequence case, its phase (I+II) and phase(III+IV) respectively are59and80, while or (5/5+5/6+6/6), they respectively are61and41and P=0.008. For (7/X+8/X) repetitive sequence case, its death case and survival caserespectively are60and79, while for (5/5+5/6+6/6), they respectively are25and77andP=0.003. For (7/X+8/X) repetitive sequence case, its reoccurrence case and transfer caserespectively are86and53, while for (5/5+5/6+6/6), they respectively are42and60andP=0.002. For (7/X+8/X) repetitive sequence case, its average survival time is15.73±7.68months, while for (5/5+5/6+6/6), it is19.46±7.67months and P=0.000.Conclusion:1. XRCC1-280His is a high risk genotype for people with positive HBV surfaceantigen or the family history of liver cancer to be attacked by liver cancer.2. XPD-312Asn is a high risk genotype for people with the habits of smoking anddrinking, carrying HBV or with the family history of liver cancer to be attacked by livercancer.3. Gene XRCC1-194Trp is not the susceptibility gene of liver cancer for Han peopleliving in southwestern China.4. The combination of gene XRCC1-194Trp+XPD-312Asn and XRCC1-280His+XPD-312Asn increases the HCC occurrence risk for Han people living in southwesternChina.5. For MIF－794CATT5-8gene promoter, the liver cancer patient with low repetitivesequence (5/5+5/6+6/6) genotype has properly good prognosis, while the patient with highrepetitive sequence (7/X+8/X) has comparative poor prognosis. 6. The microsatellite repetitive sequence polymorphism of MIF－794CATT genepromoter provides possibilities for prognosis judgment of liver cancer operation.