The Study Of HB-EGF On The Migration Of SMMC7721 Cells And Association Of TNFR2 Gene Polymorphisms With Susceptibility To HBV-Related Liver Disease

Objective::1. To evaluated the possible roles of HB-EGF in cell migration of HCC cells, and whether FAK is involved in HB-EGF induced HCC cells migration.2. To investigate the value of TNFR2 gene, rs 1061622 and rs 1061624 SNP for the susceptibility to HBV-related diseases risk in Guangxi Zhuang populations.Methods:1. Transwell Plate was used to evaluate the effects of HB-EGF on the migration of SMMC7721 in vitro. Western bolt analyses were applied to evaluate the effect of HB-EGF on the expression of FAK and phospho rylated FAK (p-FAK).2. A total of 115 patients with chronic hepatitis B (CHB),86 patients with HBV-related liver cirrhosis (LC),272 patients with hepatitis B virus (HBV)-related HCC and 269 healthy controls were subjected to a case-control study. The functional polymorphism of TNFR2 was genotyped by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for screening, followed by nucleotide sequencing for confirmation. Statistics were performed using SPSS 16.0 software. Demographic and clinical data between groups were compared by one-way ANOVA test for categorical variables and by χ2 test for continuous variables. Chi-analysis or the Fisher’s exact probability test (when the expected number in any cell was less than five) was utilized to evaluate the association of the TNFR1B genotypes d allelic frequencies in the case-control populations.Results:1. HB-EGF is a chemotactic factor in SMMC7721; HB-EGF increased FAK and p-FAK expression, FAK inhibitor 4 significantly inhibited both intrinsic and HB-EGF induced cell migration.2. There were three genotypes in TNFR2 rs 1061622 polymorphism (TT, TG and GG). The difference of the genotype and allele frequency distribution between the groups of patients and healthy controls were no statistically significant. There were three genotypes in TNFR2 rs 1061624 polymorphism (GG, GA and AA). The AA genotype was associated with increased risk of CHB and LC (P=0.026, OR=2.666,95%CI=1.126-6.316; P=0.027, OR=2.947, 95%CI=1.130-7.689); the GA genotype was associated with a significant increased HCC risk as compared with the GG genotype (P=0.046, OR1.563, 95%CI=1.009-2.422)3. When stratified by gender, the rs 1061624 site AA genotype was associated with a significantly increased risk of CHB and LC compared with GG genotype in males (P=0.011, OR=4.280,95%CI=1.405-13.041; P=0.031, OR=3.361,95%CI=1.114-10.146). the A allele was associated with increased risk of CHB(P=0.028, OR=1.668,95%CI=1.056-2.634).4. When stratified by age, the AA genotype of TNFR2 rs 1061622 polymorphism was associated with significantly increased risk of CHB and LC in people less than 50 years old (P=0.029, OR=3.000,95%CI=1.122-8.027; P=0.017, OR=5.694,95%CI= 1.019-7.216), GA genotype was associated with a significant increased LC risk (P=0.046, OR=2.712,95%CI=1.019-7.216). The A allele was associated with increased risk of LC(P=0.036, OR=1.719, 95%CI=1.036-2.854).5. When stratified by smoking, the rs 1061624 site AA genotype was associated with a significantly increased risk of LC compared with GG genotype in non-smokers (P=0.032, OR=3.855,95% CI=1.122-13.239).6. When stratified by drinking, the rs 1061624 site AA genotype was associated with a significantly increased risk of CHB compared with GG genotype in alcoholics (P=0.0.033, OR=6.096,95%CI=1.162-31.974).7. In the Han population, the rs 1061622 site A allele increase the risk of HCC (P=0.044, OR=1.497,95%CI=1.011-2.216). AA genotype increase the risk of HCC and CHB(P=0.021, OR=3.969,95%CI=1.235-12.752; P=0.031, OR=3.390,95%CI=1.120-10.26). GA genotype increase the risk of HCC and LC (P=0.035, OR=1.975,95%CI=1.048-3.722;P=0.034, OR=2.706, 95%CI=1.077-6.800).Conclusion:1. FAK is involved in HB-EGFe induced SMMC7721 migration.2. TNFR2 rs 1061622 polymorphism was not associated with HBV-related hepatitis, liver cirrhosis and hepatocelluar carcinoma risk.3. AA genotype of rs 1061624 polymorphism was associated with CHB and LC susceptibility, and it might be a risk factor for CHB and LC.4. AA genotype of rs 1061624 polymorphism was a risk factor for CHB in male group, alcoholics group and those younger than 50 years old, and for LC in male group, Han nationality group, non-smoking group and those younger than 50 years old, and for HCC in Han nationality group. GA genotype might be a risk factor for LC in people younger than 50 years old.