The Association Of KAT2B Gene Polymorphism With Clinico-pathological Characteristics Of Hepatocellular And Its Biological Function Research

ObjectiveKAT2B is a related gene on the cell cycle pathway and plays a role in acetylation and transcriptional regulation for many biological processes.The encoded protein also has the activity of acetyltransferase.Studies have shown that KAT2B is associated with gastric cancer,colorectal cancer,breast cancer,lung cancer and liver cancer.The results of our previous study showed that after adjusting for age,sex,nation,smoking,drinking,chronic HBV infection and HCC family history,KAT2B rs17006625 polymorphism was associated with the risk of HCC(P=0.04,OR=1.64,OR95%CI:1.01-2.64).This study was conducted to investigate the association between KAT2B rs17006625 and HCC clinic-pathologicalcharacteristic,theeffectofKAT2Brs17006625polymorphism on the biological function of hepatocellular carcinoma(HCC)cell line.Methods(1)498 new HCC patients who were diagnosed according to unified diagnostic criteria were selected and genetyped by Taq MGB real-time fluorescent quantitative polymerase chain reaction(RT-PCR).The generalized demographic imformation,past medical history,personal history,family history,HBV infection history,biochemical and immunological imformation,tumor size,number of tumors,cirrhosis,satellite tumor nodule,tumor thrombus,pseudocapsule,capsule and blood vessels invasion,Edmondson-Steiner grade and other clinic-pathological information were collected by unified questionnaires.SPSS17.0 software was used to analyze the correlation between KAT2B rs17006625 and HCC clinic-pathological characteristic.(2)Through bioinformatics to find the sequence of KAT2B,amplified the target gene fragment by cDNA from human Hep G2 cell mRNA,constructed vector containing vacant,wild and mutant plasmid of KAT2B rs17006625,and transfected into human HepG2 cells to obtain the hepatoma cell lines containing three different plasmids.The expression of KAT2B gene was detected by dual-luciferase experiment,real-time quantitative PCR and western blot.The ability to migration,invasion and proliferation of hepatocellular carcinoma cell were detected by transwell and MTT assay.Results(1)Association of genetic polymorphism and HCC clinic-pathological characteristic:After adjusting for gender,age,nation,HCC family history,smoking history,drinking history and HBs Ag by multivariable logistic regression analysis,the results of analysis showed that compared with the genotype AA,KAT2B rs17006625 genotype GG patients suffering from capsule invasion risk was 3.267 times(P=0.017,OR=3.267,OR95%CI:1.241-8.597),and suffering from poorly differentiated tumors risk was 3.417 times(P=0.049,OR=3.417,OR95%CI:1.003-11.637).(2)Stratified analysis:We conducted a stratified analysis based on HCC family history,smoking,drinking and HBV infection.In patients without HCC family history,KAT2B rs17006625 was associated with pathological grading of HCC(P=0.025,OR=4.555,OR95%CI:1.215-17.083).In patients with smoking,drinking and HBsAg-negative,KAR2B rs17006625 was associated with the capsule invasion risk(P<0.05).(3)Dual-luciferase experiment:In the hepatoma cell lines containing vacant,wild and mutant plasmid of KAT2B rs17006625,the relative luciferase activity values from high to low in turn was the vacant plasmid group(3.90±0.23),the wild plasmid group(3.10±0.63)and the mutant plasmid group(2.96±0.34).But there was no statistical significant between the wild plasmid group and the mutant plasmid group(P=0.747).(4)Real-time quantitative PCR:Taked the 2~-??Ct??Ct value of the first group of wild plasmid group as 1,the results of the experiment showed that the mRNA level of KAT2B in the mutant plasmid group was higher than that in the wild plasmid group(P=0.015).(5)Western Blot experiment:The expression of KAT2B protein in the hepatoma cell lines containing vacant plasmid,wild plasmid and mutant plasmid was detected by Western Blot experiment which taked the?-actin as the internal reference.The results showed that the expression of KAT2B protien in the mutant plasmid group was upregulated compared with the wild plasmid group.(6)Transwell cell migration experiment:Through staining of microscopy and microscopic visual field cell counting,the results showed that there was no statistical significant in the migration ability between the mutant plasmid group and the wild plasmid group(P=0.736,P=0.191).(7)Transwell cell invasion experiment:Through staining of microscopy and microscopic visual field cell counting,the results showed that the invasive ability of KAT2B rs17006625 mutant plasmid group was higher than that of wild plasmid group(P=0.018,P=0.003).(8)MTT experiment:The OD450nm values of the hepatoma cell lines containing vacant plasmid,wild plasmid and mutant plasmid were detected by MTT experiment.The results showed that there was no statistical significant in three hepatoma cell lines which were cultured for 24h,48h and 72h respectively(P=0.097).Conclusion(1)The G allele genotype of KAT2B rs17006625 may affect capsular invasion and Edmondson-Steiner grade of hepatocellular carcinoma.(2)The G allele genotype of KAT2B rs17006625 may increase the gene express and invasive ability of hepatocellular carcinoma cell.