| Objective: To study the mutation of TP53(P53),CTNNB1 and CDKN2A(P16)gene in HBV-related hepatocellular carcinoma and the relationship between the protein expression and clinicopathological characteristics in Fujian Province,in order to find out whether they have the specific molecular diagnostic site;To study the characteristics of HBV genotypes and to establish the epidemiological data of HBV-related HCC in Fujian Province and to explore the relationship between the genotype of HBV and HCC from the perspective of molecular biology,so as to lay the foundation for the discovery and diagnosis and comprehensive treatment of HCC in the early stage.Methods:1.130 cases of HBV-related HCC and adjacent nontumorous tissues and 30 cases of HCC without HBV infection who were hospitalized for radical resection of HCC in Fujian Province were detected the mutation of exon 5、6、7、8 of P53、exon 3 of CTNNB1 and exon 1、2、3 of P16 by PCR combined with direct sequencing,and to analyze the abnormal change of gene and its clinicopathological significance.2.IHC was used to detect the expression of p53、CTNNB1 and p16 protein in HBV-related HCC tissues,and to analyze its clinical pathological significance.3.The Type-Specific Nested-PCR was used to detect the HBV genotype in HBVrelated HCC,and to analyze the epidemiology of HBV genotyping and the relationship between HBV and the mutation and expression of P53 gene.Results:1.Exon of P53 gene sequencing results showed that the mutation of P53 gene was found in 52 cases(17.9%),50 cases in HBV-related HCC and 2 cases in HCC without HBV infection.The mutation rate of HBV-related HCC(38.4%) was significantly higher than HCC without HBV infection(6.6%)(P<0.05).The mutation rate of Codon 249 in exon 7 was highest and it was found in 35cases(AGG→AGT in 28 cases,AGG→ATG in 7 cases),34 cases in HBVrelated HCC and 1 cases in HCC without HBV infection.Besides,Codon 259 in exon 7 changed GAC → AAC in 8 cases,Codon 258 in exon 7 changed GAA→GAT in 3 cases,Codon 248 in exon 7 changed CGG→CAG in 3 cases,Codon 251 in exon 7 changed ATC→AAC in 1 cases,Codon 244 in exon 7changed GGC → GGT in 1 cases,The number of codon 259 mutations was higher than that of other related reports,and 244 codon mutations which have not been reported in China.Codon 132 in exon 5 changed AAG→AAT in 1cases.Mutations were not detected in P53 exon6 and P53 exon8.To analyzed the relationship between P53 exon7 mutation and clinicopathological parameters,we found that the mutation rate in middle and low differentiated HCC tissues was higher than that in high differentiated ones(P<0.05)..2.Exon of CTNNB1 gene sequencing results showed that the mutation of CTNNB1 gene was found in 11 cases(3.7%)in HBV-related HCC.But mutation of adjacent nontumorous tissues and HCC without HBV infection were not founded.The mutation rate of Codon 37 in exon 3 was highest and it was found in 9 cases(TCT → TAT),Besides,Codon 35 in exon 2 changed ATC→AAC in 2 cases.3.Exon of P16 gene sequencing results showed that the mutation of P16 gene was found in 2 cases(0.7%)in HBV-related HCC.But the mutation of adjacent nontumorous tissues and HCC without HBV infection were not founded.Codon 104 in exon 1 changed GGC→TGC in 1 cases,Codon 251 in exon 3 changed GGG→CGG in 1 cases.Mutations were not detected in P16exon2.4.Exon of P16 gene were detected exon deletion by PCR amplification,including 13 cases(4.4%)with deletion of exon1,51 cases(17.5%)with deletion of exon2 and 1 cases(0.3%)with deletion of exon3.The deletion of P16 gene exon1、exon 2 in HBV-related HCC was significantly higher than adjacent nontumorous tissues and HCC without HBV infection(P<0.05).5.The positive rate of p53 was 56.1%(73/130)in HBV-related HCC was higher than the non-tumorous adjacent tissues 13%(17/130)(P<0.05),but had no significant difference in HCC without HBV infection(P >0.05).The high expression of p53 was significant in recurrence 、 intravascular cancer embolus and Edmondson degree(P<0.05),recurrent cancer with intravascular tumor thrombus and middle and low differentiated patients was higher than others;The positive rate of β-catenin was 58.4%(76/130)in HBV-related HCC was higher than the non-tumorous adjacent tissues 18.4%(24/130)(P<0.05),but had no significant difference in HCC without HBV infection(P>0.05).The high expression of β-catenin was significant in recurrence 、intravascular cancer embolus、Edmondson degree(P<0.05),recurrent cancer with intravascular tumor thrombus and middle and low differentiated patients was higher than others;The positive rate of p16 was 49.2%(64/130)in HBV-related HCC was lower than the non-tumorous adjacent tissues 70%(91/130)(P<0.05),but had no significant difference in HCC without HBV infection(P > 0.05).The high expression of p16 was significant in recurrence、Edmondson degree(P<0.05),recurrent cancer with middle and low differentiated patients was higher than others.6.Three HBV genotypes B 、 C 、 B/C were detected,occupying 73.8%(96/130)、22.4%(29/130)、3.8%(5/130),and have obvious differences between types(P < 0.05).There was a close relationship between HBV genotype and age、 HBV DNA viral load(P<0.05),and was no significant in cases of P53 gene mutation(P > 0.05),but the positive rate of p53 in genotypes C、B/C was higher than B(P<0.05).Conclusion:1.The mutation of P53 gene in HBV-related HCC in Fujian Province often occurred and the Codon 249 of exon 7 of P53 gene was the mutation hot spot.2.HBV infection may increase the incidence of P53 gene mutation and leading to the occurrence of HCC.3.TP53,CTNNB1 gene overexpression may be related to gene mutation,but one of the reasons for the down-regulation of P16 gene expression may be due to the deletion of P16 gene.4.The genotype of HBV in Fujian province was mainly B and C,and a small amount of B/C,among which genotype B was the largest number.5.The positive rate of p53 protein expression in C genotype was significantly higher than that of B and B/C genotype,that is,the expression of p53 protein was related to genotype. |
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