The Role Of Hepatocellular Carcinoma Associated Antigen Cdc25C In The Genesis And Development Of Hepatic Cellular Cancer

Objective: evaluate the role of Cdc25C(cell division cyclin 25 homolog C)in the genesis and development of HCC(Hepatic Cellular Cancer).Methods:(1)recombination and prokaryotic expression of Cdc25 C fusion protein.(2)Screening of Cdc25 C specific peptide and preparation of its polyclonal antibody.(3)Cdc25C protein expression in liver disease patients were analyzed using the methods of immunohistochemistry.(4)Cdc25C mRNA expression in HCC patients were analyzed using the methods of in situ nucleic acid hybridization.(5)Serum antibody to Cdc25 C was tested by ELISA method.Result:(1)After IPTG induced,the p ET-32a(+)-Cdc25 C recombinant plasmid could express TRx-His-Cdc25 C fusion protein.SDS-PAGE electrophoresis showed that the target protein was mainly existence in the form of inclusion body,and the relative molecular weight was 73 k Da.The “His” tag of Cdc25 C fusion protein did not affect its immune response in Western bloting.(2)The properties of Cdc25 C protein antigen were analyzed by bioinformatics software.Several candidate partial peptides of low homology and high antigenicity were designed based on the purity,amino acid composition,length,hydrophilic and secondary structure.Three partial peptides of Cdc25 C were synthesized.The titers of the three corresponding anti-Cdc25 C serum were1:16000,1:16000 and 1:32000,respectively.(3)Immunohistochemical method shown Cdc25 C was expressed in human normal liver tissues,viral hepatitis B tissues,cirrhosis of the liver tissues,hepatocellular carcinoma tissues and its adjacent tissues,the positive rate is 34.7%(8 / 23),70%(7 / 10),72.5%(29 / 40),70%(28 / 40)and 35%(14 / 40),respectively.The positive rate of normal liver tissue was significantly lower than that of normal liver tissue(P < 0.05).The positive staining was mainly localized in the cytoplasm of hepatocytes,and no positive staining was found in the other cells.The average optical density of Cdc25 C positive staining in HCC tissue was(0.392±0.075).The protein expression intensity was significantly higher than that in the HCC adjacent tissues and other groups.The difference was statistically significant(P<0.05).By pairwise comparison,Cdc25 C protein in normal liver tissues and HCC adjacent tissues are lower than other groups.The difference was statistically significant(P < 0.05).But,there are no statistical difference between viral hepatitis B tissues and liver cirrhosis tissues(P > 0.05).Analysis combined with clinical parameters shown that the high expression of Cdc25 C protein in HCC tissue was correlated with sex,tumor stage and liver cirrhosis(P<0.05),but not with age and tumor size(P>0.05).(4)In situ hybridization(ISH)method shown that Cdc25 C mRNA were expressed in HCC tissues and its adjacent tissues.The positive rates were 60%(24 / 40)and 37.5%(15 / 40).The positive rate of Cdc25 C mRNA in HCC tissues was significant difference with its adjacent tissues(P < 0.05).In the detection of tissues,we can see a lot tan particles for Cdc25 C mRNA which only exists in cytoplasm.The average optical density of mRNA Cdc25 C in HCC tissues and its adjacent tissues were 0.122±0.014 and0.062±0.017 respectively.The mRNA expression intensity in HCC was significantly higher than that in its adjacent tissues.The difference was statistically significant(P<0.05).Analysis combined with clinical parameters suggested that the high expression of Cdc25 C mRNA in HCC tissue is related to the tumor clinical pathologic grade and whether with cirrhosis or not(P<0.05),but it had no relationship to age,sex and tumor size.(5)Cdc25C antibody could be detected in the serum of patients with HCC,liver cirrhosis,viral hepatitis B and normal human.The positive rate was 85.2%,81.9%,77.8% and 57.3%respectively.The positive rate of Cdc25 C antibodies in the serum of patients with HCC was significantly higher than other groups(P < 0.05).By pairwise comparison,the positive rate of Cdc25 C antibody in the serum of normal persons was lower than other groups(P<0.05),and there was no significant difference between patients with cirrhosis and viral hepatitis B(P>0.05).Analysis combined with clinical parameters suggested that the positive rate of Cdc25 C antibody was related to the decrease of serum albumin(P<0.05),but it had nothing to do with age,sex,degree of jaundice and ALT value.Conclusion: Cdc25 C fusion protein was successfully induced.Cdc25 C specific peptides were screened and polyclonal antibody was prepared.Cdc25 C can induce humoral immune response,and the level of Cdc25 c mRNA expression and Cdc25 C protein expression were closely related to the occurrence and development of HCC.