| Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies in the worldwide, and has been ranked as the 2nd cancer killer in China, the incidence and the mortality have been increasing. In all the tumor suppressor genes, p53 gene has been intensively studied, and the p53 gene mutation has been detected in more than 50% of human tumors. p53 is one of the central controlling molecules in a number of important cellular processes. For example, p53 has been implicated in the control of the cell cycle, apoptosis, senescence, differentiation, DNA repair, and maintenance of genome stability. Therefore p53 gene's mutation and inactivation have become the focus of tumor-related research today.In our previous study, 11exons of p53 gene were amplified by polymerase chain reaction (PCR) in 202 Chinese HCC patients, and the mutations were detected by direct DNA sequencing in the 11 exons. 25 types of single nucleotide mutation which change the amino acid were identified in 78 cases and 1 type of synonymous mutation in one case. Our detection compared with the p53 gene mutation in Chinese HCCs study, only 2 types mutants R249S(AGG-AGT,AGG-AGC)and V157F have been reported, however the other 22 types mutants have not. In order to explore the relationship between the p53 mutation and the HCC, 4 types mutants unreported in Chinese HCC, C141Y,R156P,C176F,P191S were performed the study on cytological function. The mutation ratio of the four mutans in the study was same, 0.495 %.Firstly, the wild type p53 (wtp53) were cloned from the Human Liver cDNA Library, then the 4 types recombinants pCMV-C141Y,pCMV-R156P,pCMV-C176F,pCMV-P191S were constructed taking pCMV-p53 as the parental template by the site-directed mutation. The p53-null cell line H1299 were transfected with equal recombinant pCMV-p53,pCMV-C141Y,pCMV-R156P,pCMV-C176F and pCMV-P191S. Western Blot result indicated that the wtp53 gene and the 4 types mtp53 gene nearly have the equal protein expression level when transfected with the equal recombinant. The luciferase assay of pP53-Luc-TA and p21-promoter reporter plasmid showed that compared with that of wtp53, the relative luciferase intensity of the C141Y,R156P,C176F significantly fell, approximated to empty-plasmid pCMV, but as for the pP53-Luc-TA , P191S nearly had the equal luciferase intensity to the wtp53, and as for the p21-promoter reporter plasmid, the relative luciferase intensity of P191S was nearly 2.5 times more than the wtp53. The apoptosis assay from flow cytometry (FCM) results showed that the mutants C141Y,R156P,C176 were lack of the ability to induce apoptosis, the apoptosis rate induced by C141Y,R156P only reached to 1/3 versus to the wtp53, the apoptosis rate induced by C176F was 2/3 versus to the wtp53, however the P191S had the equal inducing apoptosis ability to the wtp53.The results from the study demonstrated that the functional difference of the 4 types p53 mutants may own to the mutation itself but not caused by the different protein expression level. C141Y,R156P,C176F mutation led to the lack of the transcriptional activity and the inducing apoptosis ability, however the P191S mutation had no significantly influence on the two cytological function, which indicate that the relationship between the p53 mutation and the tumorgenesis has the other unknown mechanism except the known one that the mutated p53 influenced transcriptional activity and the inducing apoptosis ability.The thesis firstly performed the mutation detection in p53 gene's 11 exons in 202 Chinese HCC samples, and the results provided detailed information for the global p53 gene mutation database and enriched p53 gene mutation database in the Chinese HCCs . The study on cytological function of the C141Y,R156P,C176F,P191S helped to further investigate the relationship between p53 mutation and the HCC pathological mechanism, provided valuable evidence for understanding molecular mechanism of HCCs and set up good foundation for the molecular therapy based on the p53.
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