Studies On Key Genes Of The Urea Cycle Induced By Aflatoxin B1 And The Candidate Gene Cps1

Hepatocellular carcinoma (HCC)is one of the most common malignancies,the study on HCC includeing the complexity of its pathological mechanism ,is helpful for pathogenesis,diagnosis and treatment. AFB1 is a strong agen inducing liver cancer,so it is widely used in animal model of liver cancer, using in liver cancer etiology, pathogenesis, prevention and other aspects of chemistry.In this study, mice was infected with AFB1, we detected mRNA variation of CPS1 by Fluorescence Quantitative PCR after infection, As we expected, the mRNA of cps1,otc and sir5 all decrease, which was consistent with the two-dimensional electrophoresis. In the mouse modle, mouse injecting aflatoxin B1 suffered from hyperammonemia. cps1,otc and sir5 gene were was highly expressed in the testis, lung, liver and so on.Followingly, we inserted cps1,otc and sir5 into the protokaryon expression vetor pET28a/ pET32a, and transformed them into the the host E.coli BL21 (DE3) resepectively. After induced by IPTG, the positive bacteria successfully expressed OTC and SIR5 protein. Purified the target protein was used to immunize mice, and the serum titer was detected by indirectly ELISA,prepared polyclonal antibodies. Alose, we Identified that the OTC protein interact with SIR5 by Far western Finally, we sutdied the cell biological function of CPS1. Firstly,we constructed Interference vector to cps1, and transformed them into the the host BEL-7402 and chang liver cells resepectively. In cell lines of cps1 interference,we detected that the OTC and SIR5 proteins had decreased. Next,we detected the IC50 by MTT. On this basis, MTT results showed that the Survival cells of RNAi group decreased infecting AFB1. Also,mRNA levels of the cps1 in cell lines chang liver cells were examinated by Fluorescence Quantitative PCR after infection.As we know CPS1 and OTC were key enzymes in the urea cycle to detoxify ammonium produced from amino acid catabolism. Our results may imply that aflatoxin B1 can interrupt the urea cycle and CPS1 may be involved in the development and progression of human HCC. CPS1 could be a molecular marker for the diagnosis and the prognosis analysis of HCC.