The Detection And Analysis On The ND5 F340Y Mutation Of Encoding Gene Of Mitochondrial DNA In Esophageal Squamous Carcinoma

Objective: In recent years, with increased study of mitochondria, it has been discovered that the existence of DNA in mitochondrial basilar membrane, it is a more unique genetic material that only exists outside the core. Previous research has shown that mitochondrial DNA(mt DNA) ND1 mutations maybe play certain role in the occurrence and development of Esophageal Squamous Carcinoma(ESCC). There are few domestic and international reports on the relationship in terms of functional role and energetic metabolism between the encoding gene mutation of mt DNA and esophageal squamous carcinoma. Hereby we investigate the mutations on the encoding region of mt DNA in carcinoma tissues and paired normal tissue samples, and find the high frequency mutation genes and sites, and to analyze its effects on energy metabolism esophageal squamous carcinoma.Methods: Normal esophagus tissue, and esophageal squamous carcinoma tissues were taken from the resected specimens of 30 patients with esophageal squamous carcinoma who underwent surgery in the second affiliated hospital of Zhengzhou university and the first affiliated hospital of Zhengzhou university. The tissue samples put in the negative 80 degrees freezer to be used as a backup. Extract the mt DNA from tissue samples, the mt DNA displacement encoding regions were amplified by PCR.Direct sequencing was followed,Using the NCBI databases gene sequence analysis software, compare the mt DNA encoding regions with the revised Cambridge Reference Sequence(r CRS), the high incidence mutation sites and genes were examined, use Mutation Assessor to predict the functional impacts, and use immunohistochemistry technology to analysis the energy metabolism.Result: 1. 216 mutations were identified in 30 cases of esophageal squamous carcinoma, including 175 inherited mutations, 41 somatic mutations. Among these 216 mutations, 182 were protein coding mutations(63 missense mutations).2. In 216 mutations, there were ten heterogeneity mutations, all remaining for homogeneity mutations.3. Functional impact analyses of the somatic and inherited missense mutations by using Mutation Assessor program: we find that somatic mt DNA mutations(3/12) have more influential power than germline mutations(2/46).4. Among the five high impact missense mutations, the F340 Y mutation in ND5 gene was found with the highest functional impact score(FI score=5.235) and the highest frequency(26.7%). P-AMPK Thr172 immunereactivity assay demonstrates that F340 Y mutation correlates with energetic stress condition, F340 Y mutant ESCC samples were observed with higher p AMPK immunoreactivity in comparison to the wild type samples(P<0.05).Conclusion: In the current study, five high functional impact somatic and inherited mutations were identified in ESCC patients. Among these, the F340 Y mutation was found with the highest impact score and the highest frequency. The finding that the F340 Y mutation correlates with higher energetic stress status further supports the effect of the mutation on ESCC cells' energetic metabolism. Research and analysis on high frequency mutations may be used in the early diagnosis and molecular targeting treatment of the tumor.