| Background: Hepatocellular carcinoma is one of the most commonmalignant tumors in the world, in recent years the rate of incidence andmortality keep high level. The number of new cases worldwide is600000,ranking the fifth malignant tumor,and deaths to598000cases each year. Thedeath rate of liver cancer ranked third,just following the stomach, esophaguscancer. Hepatocellular carcinoma has been a serious threat to human health andlife. Statistic data shows that half of new hepatocellular carcinoma cases anddeaths reported worldwide occur in China. Its distribution has obvious regionaldifferences. Accorrding to the epidemiological investigation, the incidence andmortality rate of liver cancer in Guangxi were significantly higher than that ofthe national average,and the liver cancer in Guangxi showed tendency offamilial aggregation. Most patients with hepatocellular carcinoma had familyhistory of liver cancer. The risk of suffering from HCC increased greatly withintheir Ⅰ and Ⅱdegree relatives. The majority of hepatocellular carcinoma presented insidious onset, rapid progress, short course, so far,there is no specifictreatments to prevent tumor, so it is necessary to conduct in-depth research onthe pathogenesis of liver cancer and familial aggregation focusing on theinternal genetic factors and environmental factors.After many years of hard work, domestic and foreign experts make someachievement in finding out the pathogenesis of liver cancer. Accorrding tothe epidemiological investigation in some areas of Guangxi with highincidence of hepatocellular carcinoma, experts agree that HCC is due to thefollowing factors: infection with chronic hepatitis B virus, intake of aflatoxinand chemical toxicant(including nitrosamines, heavy metals elements), intakeof water with algal toxins, exposure to radiation, alcohol consumption,infection by hepatotropic parasites, genetic factors and so on. But the specificmechanism of interaction among biological, perplexing environment, geneticfactors remain unclear. The cause of familial clustering of hepatocellularcarcinoma was more complex, many studies suggest that infection with hepatitisB virus was a main pathogenic factor of HBV related HCC. Our previous studyfound that HBV infection is the main reason causing familial clustering ofhepatocellular carcinoma in Guangxi population. Other factors included geneticfactors, HCV infection and environmental factors, such as water pollution oraflatoxin. No matter how the carcinogenic mechanism of biological factor,environment and genetic factors, the body will appear immune function change.A variety of immune cell function and (or) the expression of cytokines wasabnormal. The balance between anti-inflammatory factors and inflammatoryfactors was broken, the humoral immunity and cellular immunity weredysfunction, causing vicious spiral of abnormal immune response function. Forexample, The body with hepatocellular carcinoma lost balance of cytokine between Th1cytokines IL-1, IL-2, IL-12, IL-18, TNF-a, IFN-r and Th2cytokines IL-4, IL-5, IL-8, IL-10,which were belongs to inflammationpromoting factor and inflammation inhibition factor respectively.That will leadCD4+cells,CD8+cells, NK cells, cytotoxic T cells, macrophages and otherfull-time work immune cells to lower ability or abnormal function,resulting inthat tumor escaped from immune surveillance and immune clearance,and thatmake tumor produce, progress and metastasis. It means that liver cance is atypical tumor associated with inflammation at the same time. It was knownthat cytokines such as IL-18, IL-10, IL-1, IL-12, TGF-β1, TNF-α, NQo1, XPDand COX2gene polymorphism was associated with HCC. Among thesecytokines, the transforming growth factor beta1(TGF-β1) gene polymorphismplays an important role in production of primary hepatocellular carcinoma.TGF-β1is a kind of hormone like peptides to regulate cell proliferation,differentiation and function, inhibit lymphocyte, epithelial cell proliferation anddifferentiation, can promote proliferation of mesenchymal cell, regulatesynthesis of extracellular matrix proteins, plays an important role in the processof tissue repairation and cells mutation. Normal levels of TGF-β1suppressimmune function properly through inhibiting activation of T lymphocyte and Blymphocyte, inhibiting of killing function of NK cells and cytotoxic T cell.Under pathological conditions, TGF-β1level increases obviously, whichdestroys the immune surveillance function, makes the body out of the normalimmune response to foreign antigen or tumor cells, which gives chance todevelop diseases or tumor. Relationships between cytokines above and familialclustering of hepatocellular carcinoma continued to be reported, but there wasno report about the effect of genetic polymorphisms of TGF-β1on familialclustering of liver cancer recent years. The relationship between the expression of TGF-β1and familial aggregation of hepatocellular carcinoma is unclear. Inorder to learn about factors associated with familial clustering of hepatocellularcarcinoma occurred in Guangxi better, we explore the causes of familialclustering of hepatocellular carcinoma in the perspective of transforming growthfactor β1gene polymorphism and its protein expression.There are two parts ofthe study. Firstly, to obtain case group and control group genetics data of sixsites of transforming growth factor β1,we analysis this sites gene polymorphismof transforming growth factor β1,which associated with HCC, with214familymembers(including105cases of zhuang、60cases of Han and49cases of Yao)in34hepatocellular carcinoma clustering families (FHCC) and214familymembers (including105cases of zhuang、60cases of Han and49cases of Yao)from the families without any cancer (FNC), those of all that from11highincidence area of liver cancer in Guangxi population, probe the effect oftransforming growth factor β1gene polymorphism on hepatocellular carcinomaclustering families exposure to smoking, drinking, HBV infection together. Thesecond part of the study is divided into two small parts: Firstly, compare thetransforming growth factor β1level in serum between the hepatocellularcarcinoma clustering families (FHCC) group and the families without anycancer (FNC), compare serum TGF-β1levels among different groups of riskloci genotypes, analysis stratification to serum TGF-β1levels according to theblood relationship, with ELISA assay to detect the serum TGF-β1level of428cases. Second, use western blot assay to detect blood protein expression of82cases including11probands and their matches,10cases of first-degree relativesselected randomly in hepatocellular carcinoma clustering families and theirmatches,10cases of second degree relatives selected randomly inhepatocellular carcinoma clustering families and their matches,10cases of third degree relatives selected randomly in hepatocellular carcinoma clusteringfamilies and their matches, compare the difference of blood TGF-β1proteingray value between case group and control group, analysis blood TGF-β1protein gray value according to the blood relationship with stratification,explore relation of TGF-β1and liver cancer familial aggregation in perspectiveof protein expression.Part Ⅰ Study on the relationship between TGF-β1genepolymorphisms in GuangXi China population and familialclustering of hepatocellular carcinomaObjectives: To probe the susceptibility of cytokine TGF-β1genepolymorphism and exposure factors with primary hepatocellular carcinoma inGuangxi Zhuang, Han, Yao population.Methods: confirm214family members from34hepatocellular carcinomaclustering families (FHCC) as the cases and37families without any cancer(FNC),select214members from37families without any cancer (FNC) as thecontrol matched214member of the cases in terms of ages(±5), HBsAg,nationality, habitation and gender. All subjects were from11areas of highincidence rate of hepatocellular carcinoma in Guangxi population. Acquire basicinformation and liver cancer risk factors of the subjects by epidemiologicalquestionaire. Collect5ml non-anticoagulant blood to separate of serum fordetection of markers of HBV, anti-HCV, TGF-β1levels.5ml fasting venousblood of428subjects were collected in anticoagulant tube in the morning, andgenome DNA were extracted. The specific primer PCR amplified6SNPloci of TGF-β1rs1800469, rs2241715, rs2241716, rs11466345, rs8105161, rs747857, which were genotyped through the method of Snapshop. Haploviewsoftware was applied to test Hardy-Weinberg genetic balance balance, andSHEsis software was applied to analysis linkage disequilibrium and haplotypefrequency. All data were input and analyzed statistically in SPSS17.0. Chisquare test and conditional Logistic regression model were used to analysisrelationship between TGF-β1SNP and liver cancer familial aggregation.Crossover analysis for SAS9.1performed to gene-environmental interactionstudies for statistically significant Loci and environmental factors.Result:(1) Distributions of suspected risks of HCC in the subjects: thefrequency of drinking、smoking、Drinking pond water and HBV-DNA(+) weresignificant different in the members of FHCC and those of FNC.(2) Hardy Weinberg equilibrium test: the result show that all sis sitesgenotypes of the observations fit well with the theoretical value(P>0.05) byHaploview software test in hepatocellular carcinoma clustering families (FHCC)group and the families without any cancer (FNC) group, show that samples ofthis experiment with a group representative.(3) TGF-β1gene polymorphism genotypes and allele frequency in FHCCgroup and FNC group:1.the frequency of TGF-β11800469sites of genotypesTT,CT,CC were47.2%、41.1%、11.7%in FHCC group respectively,33.7%、45.3%、21．0%in FNC group respectively. There was a statistical differencebetween the FHCC group and FNC group（P=0.004）.The distribution ofTGF-β11800469of alleles T/C were67.8%and32.2%in FHCC grouprespectively,56.3%、43.7%in FNC group respectively. There was astatistical difference between the FHCC group and FNC group（P=0.001）.Thefrequency of TGF-β11800469TT and TGF-β11800469T was more higher inFHCC group than that of FNC group（P=0.004；P=0.001）.2. the frequency of TGF-β2241715sites of genotypes TT、GT、GG were46.7%、41.6%and11.7%in FHCC group respectively,33.2%、44.9%and21．9%in FNC grouprespectively. There was a statistical difference between the FHCC group andFNC group（P=0.003）.The distribution of TGF-β12241715of alleles T/Gwere67.5%and32.5%in FHCC group respectively,55.6%and44.4%inFNC group respectively. There was a statistical difference between the FHCCgroup and FNC group（P=0.000）.The frequency of TGF-β12241715TT andTGF-β12241715T was more higher in FHCC group than that of FNC group（P=0.004；P=0.000）.3. the frequency of TGF-β18105161sites of genotypesTT,CT,CC were29.4%、51.9%and18.7%in FHCC group respectively,42.5%、45.8%and11．7%in FNC group respectively. There was a statisticaldifference between the FHCC group and FNC group（P=0.009）.The distributionof TGF-β18105161of alleles T/C were55.4%and44.6%in FHCC grouprespectively,65.4%and34.6%in FNC group respectively, that was astatistical difference between the FHCC group and FNC group（P=0.003）.Thefrequency of TGF-β18105161TT genotype and TGF-β18105161T allele wasmore higher in FHCC group than that of FNC group（P=0.005；P=0.003）.4. thefrequency of TGF-β1747857site of genotypes AA,AG and GG were82.7%、15.0%and2.3%in FHCC group respectively,90.7%、9.3%and0%in FNCgroup respectively. There was a statistical difference between the FHCC groupand FNC group（P=0.005）.The distribution of TGF-β1747857of alleles G/Awere90.2%and9.8%in FHCC group respectively,95.3%and4.7%inFNC group respectively, that was a statistical difference between the FHCCgroup and FNC group（P=0.004）.The frequency of TGF-β1747857GG genotypeand TGF-β1747857G allele was more higher in FHCC group than that of FNCgroup（P=0.016；P=0.004）.5.There were no statistical differences between theFHCC group and FNC group in the frequencies of the genotypes CC/TC/TT and alleles T/C at the rs2241716locus（P=0.105；P=0.160）.6. The distributionof the genotypes GG/GA/AA at the rs11466345site was no significantdifference between the cases group and the control group（P=0.161）,while thefrequency of rs11466345T alleles in the FHCC group were high than that ofFNC group（P=0.049）.(4) TGF-β1polymorphisms in linkage disequilibrium test: three sites ofrs2241715、 rs2241716and rs1800469exist in strong linkage disequilibrium.The strength of linkage showed D‘=1，r2=0.984between rs1800469andrs2241715. The two sites of rs1800469and rs2241716exist in linkagedisequilibrium(D‘=1， r2=0.547).Also, the sites of TGF-β1rs2241715andTGF-β1rs2241716showed linkage disequilibrium(D‘=1，r2=0.538).(5) TGF-β1haplotypes analysis:SHSsis software was applied to performhaplotyping analysis. CGC、CGT and TTC were the most common haplotypesin rs1800469、rs2241715、rs2241716among the two groups. The frequency ofthem was more than30%. The distribution of haplotype CGC、CGT and TTChad no significant difference among the two groups（all P>0.05）. The frequencyof FHCC group was11.2%、23.3%and64.8%，as compared with12.6%、19.6%and67.9%in the FNC group. TGC only distributed in the FNC group, thefrequency was0.7%.(6) Conditional logistic regression analysis was applied to perform multiplefactors analysis of genetic and environmental factors: list rs1800469、rs2241715、rs8105161、rs747857、rs2241716、rs11466345and the exposurefactors of smoking、drinking、drinking pond water、HBV infection intoConditional logistic regression model, lastly, the risk factors entered the modelthat were sorted in ascending order were rs2241715TT, rs1800469TT,HBV-DNA positive, alcohol consumption and drinking pond water.(7)Stratification analysis of risk genotypes: the result showed that the frequency of rs1800469TT genotype and rs1800469T allele decreased with thealienation of consanguinity, although with no statistical difference in the ratesbetween FHCC group and FNC group.(8) Application of crossover analysis on interaction of gene andenvironment: In additive model, OR value of interactive effect ofTGF-β1rs1800469TT and drinking of pool water was6.44，S=1.50，AP=0.28;OR value of interactive effect of TGF-β1rs1800469TT and drinking was3.45，S=1.29，AP=0.16;OR value of interactive effect of TGF-β1rs1800469TT andHBV-DNA was5.38，S=4.13，AP=0.62.All the interactions mentioned abovewere statistically significant(P<0.05). The assumption test U=0.394,0.432,1.105respectively, and all p<0.05.Conclusion:(1)TGF-β1rs1800469and TGF-β1rs2241715SNPs may begenetic factors of HCC familial clustering in Guangxi population.(2) TGF-β1rs8105161TT and TGF-β1rs747857GG may be protectivefactors for HCC familial clustering in Guangxi population.(3)Drinking of pond water、alcohol consumption and HBV-DNA positivemay be main exposure factors of HCC familial clustering in Guangxipopulation.(4) Composite factors caused HCC familial clustering in Guangxipopulation, multiple genetic factors, environmental factors or gene-environmentfactors exist a interaction effect. Part Ⅱ Study on the relationship between TGF-β1expressionin GuangXi China population and familial clustering ofhepatocellular carcinomaObjectives: To approach the effect of expression of TGF-β1level in serumand plasma on HCC familial clustering in Guangxi population qualitatively andquantitatively.Methods: confirm214family members from34hepatocellularcarcinoma clustering families (FHCC) as the cases and37families without anycancer (FNC),select214members from37families without any cancer (FNC)as the control matched214member of the cases in terms of ages(±5), HBsAg,nationality, habitation and gender. All subjects were from11areas in Guangxiwhere the incidence of hepatocellular carcinoma keep high level. The study isdivided into two small parts:Firstly, compare the transforming growth factor β1level in serum between the hepatocellular carcinoma clustering families (FHCC)group and the families without any cancer (FNC), compare serum TGF-β1levels among different groups of risk loci genotypes, analysis stratification toserum TGF-β1levels according to the blood relationship, with ELISA assay todetect the serum TGF-β1level of428cases. Second, use western blot assay todetect blood protein expression of82cases including11probands and theirmatches,10cases of first-degree relatives selected randomly in hepatocellularcarcinoma clustering families and their matches,10cases of second degreerelatives selected randomly in hepatocellular carcinoma clustering families andtheir matches,10cases of third degree relatives selected randomly inhepatocellular carcinoma clustering families and their matches, compare thedifference of plasma TGF-β1protein gray value between case group and control group, analysis blood TGF-β1protein gray value according to the bloodrelationship with stratification, explore the relationship of TGF-β1and livercancer familial aggregation in perspective of protein expression. Datas wereinput and analyzed statistically in SPSS17.0.Variable values can be summarisedas a mean value and standard deviation. Paired-sample t-test was used tocompare mean value of the sample within two groups. Use Kruskal-Wallis t-testto compare population distribution among multiple groups, withα=0.05P＜0.05as statistically significant.Result：1．The effect of serum TGF-β1level on HCC familial clustering(1)The serum TGF-β1level in HCC familial clustering group was muchhigher than that in FNC group.（28.495±17.495ng/mlVS20.24±7.56ng/ml，P=0.000）。(2)Stratification analysis on serum TGF-β1level according to genotype:Serum concentrations of TGF-b1were significantly higher in all rs1800469TTsubjects than that in all rs1800469CT subjects （39.445±7.445ng/mlVS26.33±13.65ng/ml， P=0.000） and all rs1800469CC subjects（39.445±7.445ng/mlVS26.245±13.555ng/ml，P=0.000）.no significantdifferences in serum TGF-b1levels were found in CT subjects with CC subjects（26.33±13.65ng/mlVS26.245±13.555，P=0.347）. Serum concentrations ofTGF-b1were significantly higher in all rs2241715TT subjects than that in allrs2241715GT subjects（30.33±15.66ng/mlVS23.55±11.45ng/ml，P=0.000）and all rs2241715GG subjects （30.33±15.66ng/mlVS20±10ng/ml， P=0.000）.no significant differences in serum TGF-b1levels were found in GTsubjects with GG subjects（23.55±11.45ng/mlVS20±10ng/ml，P=0.061）.(3) Compare of TGF-b1serum concentrations in subjects of FHCC groupand FNC group with risk genotypes: Serum concentrations of TGF-b1were significantly higher in rs1800469TT subjects in FHCC group than that inrs1800469TT subjects in FNC group（43.395±3.495ng/mlVS35.95±3.95ng/ml，P=0.000）. Serum concentrations of TGF-b1were significantly higher inrs2241715TT subjects in FHCC group than that in rs2241715TT subjects inFNC group（18.815±4.145ng/mlVS38.995±6.995ng/ml，P=0.000）.2. Results of Western blot test(1) All subjects in the case and the control group express TGF-β1proteinand no abnormal protein bands were found, but the expression of protein levelswere obviously different.(2) Compared to the FNC group, TGF-β1protein content of FHCC groupwas higher.The difference had statistically significant（p＜0.01）.(3) Stratification analysis showed that TGF-β1protein content had nostatistically difference among probands、first degree、second degree、threedegree relatives(P>0.05).TGF-β1protein gray value very high in probands, butit decreased with blood relationship.Conclusion:(1)High serum TGF-β1levels may be associated withfamilial clustering of hepatocellular carcinoma.(2)High levels of plasma TGF-β1may be associated with familial clustering of hepatocellular carcinoma.(3)High serum TGF-β1levels or high levels of plasma TGF-β1may be associatedwith immune status, But it need to be confirmed through T cell or B cellfunction. |
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