Structure Analysis Of P53 Hot Spot Mutation In Human Hepatocellular Carcinoma Non - Operative Patients With Human Autophagic Labeling Atg8 Family Protein Variation Splicing

HCC pathogenesis involves the mutation of tumor-suppressor genes or oncogenes. These mutated DNA fragments could be released to the blood as circulating-cell free DNA (cfDNA). By isolation and detection of cfDNA, we could know the genetic changes of tumors without surgery. Mutations in Tp53 are reported to be associated with prognosis of many cancers, and p53 Arg249Ser (AGG→AGT) is prevalent in China and sub-Saharan Africa. In this study, we designed and compared 3 methods to detect the p53 R249S mutation in cfDNA from HCC patients, including direct sequencing of PCR product, HRM (high resolution melting curve analysis) and ASB-realtime PCR (Allele specific block realtime PCR). At last, we chose the most sensitive (10 copies per reaction) and specific (0.1% mutation templates in wild type templates) ASB-realtime PCR to detect 569 cfDNA samples of HCC patients (421 samples were collected from non-operation patients and 148 samples were from operated patients) from Qidong, Jiangsu Province, in which we found 290 samples (51.0%) contained p53 R249S mutation. Then we found that HCC patients with p53 R249S mutation had a shorter overall survival time compared with patients with R249-wild-type p53 (hazard ratio [HR],1.933; 95% confidence interval [CI]: 1.591-2.349, P<0.0001), and the median survival time of p53 R249S patients were 7 months (95%CI:5.6-8.4) while 27 months (95%CI:18.6-35.4) that of wild-type patients (P<0.00001). The median survival time of non-operation patients who had p53 R249S mutation were 6.0 months while that of R249 wild type patiants were 16.0 months. The median survival time of operated patients who had p53 R249S mutation were 21.0 months while that of R249 wild type patiants were 61.0 months. We also found the high amount of p53 fragments (HR,1.892; 95% CI:1.558-2.296, P<0.0001) but not p53 R249S fragments in cfDNA were associated with reduced overall survival time. These results suggest that ASB-realtime PCR is a sensitive and specific tool to detect mutations in cfDNA and p53 R249S mutation is an important prognostic marker in HCC.Atg8 and its homologs have been proved to play important roles in autophagy, a conserved cellular process in eukaryotes to degrade cytoplasmic components via lysosome pathway. Human Atg8 homologs include MAP1LC3A, MAP1LC3B, MAP1LC3C (LC3A, LC3B, LC3C for short), GABARAP, GABARAPL1, and GABARAPL2. It is not clear which amino acids or damains may play important roles in the protein interaction and autophagy process, and alternative splicing analysis could provide useful information to solve these problems.We analyzed the alternative splicing patterns of human Atg8 homologs. Then we focused on LC3B-b, the new isoform of LC3B. It is formed by NAGNAG alternative splicing and resulted in the absence of Arg68. In the former results of MD simulations, we have found LC3B-b may prossess a different solution conformation. Temperature increasing CD analysis and trypsin digestion showed that LC3B-b had weaker intramolecular interaction and possessed less compact conformation. This conformational change may severly inhibit its C-terminal cleavage by cysteine protease ATG4B, the ubiquitin-like modification and autophagosome localization. Next, when Arg68 was mutated to Alanine, the pro-form of LC3B significantly increased, with fewer autophagosome localization and lower autophagic flux. This was also found in other human Atg8 homologs. Furthermore, by structure analysis, we found Arg68 of LC3B could form a salt bridge with Aspl71 of ATG4B and the mutation of Arg68 of LC3B-a or Aspl71 of ATG4B would weaken their association and reduce their cleavage efficiency. These results indicated that Arg68 was a key amino acid for the autophagic function of human Atg8 homologs.To explore the potential biological relevance of LC3B-b, we measured the mRNA expression ratio of LC3B-b and LC3B-a by qPCR and found that the expression ratio was tissue and cell line specific. We also found that the expression ratio in hepatocarcinoma cell line Hep3B would vary under starvation and hypoxia which suggested the NAGNAG splicing of LC3B might be under regulation. Further more, we selected two genes which contained the same NAGNAG motif with LC3B, and found that their splicing ratios changed differently with LC3B under starvation and hypoxia, suggesting the NAGNAG regulation was gene specific. Somehow, the biological relevance was still not clear and needed to be further studied.