Association Study Of Gene Polymorphisms And Expression Of TNF-α, TAP With Hepatocellular Carcinoma

Chronic infection of HBV is an important factor leading to different clinical outcomes including chronic hepatitis B, hepatitis B cirrhosis and hepatocellular carcinoma (HCC). Each year, about 1.2 million people worldwide die from chronic HBV infection-related liver disease. It is high-endemic area of hepatitis B virus infection in China,where there is up to 9.09% of chronic HBV infection among crowd accounting for over 1/3 of the global total.Each year it occurs in patients with hepatitis B cirrhosis and hepatocellular carcinoma (HCC) accounting for 2% and 1% among patients with chronic hepatitis B,respectively. Chronic HBV infection is threatening to people,s health more and more. Therefore, the study about the chronic mechanism of HBV infection is still an important topic in the world. The chronic mechanism of HBV infection is quitely complex, involving the combined effects of a host, viruses, environment and other factors, the combination of host immune and genetic factors may play an important role in the occurrence and progression of chronic HBV infection.lt has been payed more attention to antigen-presenting system and cytokines, because they are involved in immune response to HBV infection, and may be associated with the susceptibility to HBV infection and the clinical outcomes of infection. In which the effects of polymorphisms of TNF-α(tumour necrosis factor-α)as an important human immune regulator and TAP (transporter associated with antigen processing) as an important molecular of MHC-Ⅰrestricted antigen processing pathway on the clinical outcomes of chronic HBV infection,and expression of TNF-α,TAP1 on hepatocellular carcinoma had been studied little.The study aimed to clarify the correlation between the gene polymorphisms of TNF-α,TAP and clinical outcomes of chronic HBV infection ,and the association between expression of TNF-α,TAP1 and hepatocellular carcinoma due to HBV in a Han population in northeasten China .Objective:The aim of this study was to explore the potential relationship between polymorphisms of TNF-α,TAP and different clinical outcomes of chronic HBV infection,and the association of expression of TNF-α,TAP1 with occurrence and development of hepatocellular carcinoma due to HBV in a Han population in northeastern China. It is expected that results of the studies will be theoretical importance in exploring chronic mechanism of HBV infection and helpful to providing new indicators for diagnosis and prognosis ,and theoretical basis for the immune and gene therapy of chronic HBV infection-related diseases.Methods:1.189 HBV spontaneously recovered subjects (SR), 571 HBV chronically infected subjects including 180 chronic HB patients (CHB), 196 HB cirrhosis patients (HC) and 195 hepatacellular carcinoma patients due to HB(HCC) were included in this study. TNF-α-857 C/T and-863 C/A , TAP1-333 Ile/Val and-637 Asp/Gly ,TAP2-651 Arg/Cys and -687 Stop/Gln were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), The results were analyzed with gel imaging analytical system after agarose gel electrophoresis, and clarified the association of TNF-αgenotypes and alleles at position 857,863 , TAP alleles and four locus haplotype composed of TAP1-333,-637 and TAP2-651, -687 with chronic hepatitis B, HB cirrhosis,and hepatacellular carcinoma duo to HB by the method of case-control study.2.The expression of TNF-αand TAP1 in 35 liver carcinoma tissues, and 10 nomal liver tissues obtained through operation were detected with EnVision method belonged to immunohistochemical technique and compared with each other in order to clarify the expression and their significance of TNF-α,TAP1 in HCC due to HBV. Furthermore, the association with pathological typing was analyzed.Results:1.TNF-α-857C allele frequency was significantly higher in chronically HBV-infected individuals (CHB and HC) compared with that of SR subjects (P=0.01, OR=1.52,95%CI=1.08-2.13;P=0.02,OR=1.47,95%CI=1.06-2.04,respectively);2.TNF-α-863A allele frequency was significantly more common in HBV-infected individuals with chronic HBV infection (CHB, HC and HCC) than that of SR controls(P=0.004, OR=1.72, 95%CI=1.19-2.50;P=0.001, OR=1.81, 95%CI=1.26-2.61 and P<0.001, OR=1.90, 95%CI= 1.33-2.73, respectively).3. TAP1-637-Gly allele frequency was significantly more common in HBV-infected individuals with chronic HBV infection (CHB and HC) than that of SR controls(OR = 1.53, 95%CI1.05-2.23, P=0.026; OR =1.73, 95%CI1.20-2.48,P = 0.003);4. a statistically significant difference in the distribution of TAP2-651-Cys allele between HCC cases and SR controls(OR=2.24,95%CI1.45-3.48,P<0.001) was observed; 5.TAP2-687-Gln allele frequencie in CHB group was higher than that in SR group (OR=1.36,95% CI1.02-1.82,P=0.039); 6.Haplotype analysis also revealed that TAP haplotype [-687C;-651T; -637G; -333A] was strongly associated with chronic HBV infection (CHB, HC and HCC) compared with SR(P<0.001, P<0.05, P<0.001,respectively);7.The expression of TNF-α,TAP1 was negative in hepatocytes of normal liver tissues. 60% of HCC tissues expressed TNF-αwhich distributed in cytoplasms dispersively, There was significant difference between HCC and the normal hepatic tissues in expression of TNF-α( P<0.05),and the positive expression rate of TNF-αin well-differenciated HCC tissues was significantly higher than that in poorly-differenciated ones (P<0.05),which showed that the overexpression of TNF-αwas associated with the occurrence and development of HCC due to HB.91.4% of HCC tissues expressed TAP1 which distributed in cytoplasms intensively or dispersively, There was statistical difference in expression of TAP1 between HCC tissues and the normal hepatic tissues (P<0.05),which showed that TAP1-related MHC-Ⅰrestricted antigen processing pathway was possibly normal in HCC related to HB.Conclusion:In a Han population in northeastern China. 1.TNF-α-857C allele was close associated with CHB and HC, TNF-α-863A allele was close associated with CHB,HC and HCC. TNF-αalleles may be close associated with chronicity of HBV infection 2.TAP1-637-Gly allele was associated with CHB and HC; TAP2-651-Cys allele was associated with HCC;TAP2-687-Gln allele was associated with CHB; 3.TAP haplotype [-687C;-651T; -637G; -333A] was strongly associated with chronic HBV infection (CHB, HC and HCC),respectively;4.The expression quantity of TNF-αwas associated with occurrence and development of HCC due to HB;4.TAP1-related MHC-Ⅰrestricted antigen processing pathway is possibly normal in HCC related to HB.Compared with similar studies in China and worldwide,our research deserves to be studied and explored further which provided laboratory evidence for immune genetic mechanism of chronic HBV infection from gene and molecular level. The innovations of this research were as follows: It was the first study in Han population of northeastern China that:①TNF-αallele polymorphism was associated with chronic HBV infection including chronic hepatitis B, posthepatitis B hepatic cirrhosis and posthepatitis B hepatocellular carcinoma;②TAP allele and haplotype polymorphisms were associated with chronic HBV infection including chronic hepatitis B, posthepatitis B hepatic cirrhosis and posthepatitis B hepatocellular carcinoma;③The expression quantity of TNF-αwas associated with the occurrence and degree of pathological differentiation of HCC duo to HBV;④The strongly positive expression of TAP1 suggested that TAP1-related MHC-Ⅰrestricted antigen processing pathway is possibly normal in HCC related to HB.In summary,this study has illuminated the relationship between TNF-α,TAP alleles and TAP haplotype and different clinical outcomes of chronic HBV infection including chronic hepatitis B,posthepatitis B hepatic cirrhosis and posthepatitis B hepatocellular carcinoma in Han population in northeastern China for the first time.The correlation between gene polymorphisms of TNF-α,TAP and chronicity of HBV infection was established .we also clarified the association between the expression quantity of TNF-αand the occurrence and development of HCC related to HB,further found TAP1-related MHC-Ⅰrestricted antigen processing pathway was possibly normal in HCC related to HB by detecting the expression of TNF-αand TAP1 of liver carcinoma tissues through operation using immunohistochemical technique.our study has elucidated the mechanism of the chronicity of HBV infection.and found genetic markers of the new specific forecast for incidence of chronic HBV infection in different parts of our country, provided a theoretical basis for the immune and gene therapy of diseases from chronic HBV infection and new indicators for early disease diagnosis and prognosis of chronic HBV infection.