Study Of Relationship Between Impaired MtDNA And Gastric Carcinogenesis Plus Its Pathogenesis

Gastric carcinoma is one of the most common cancers in the world. It has been known that gastric carcinogenesis is associated with some oncogenes and tumor suppressor genes, but no definite mechanism is understood. mtDNA ( mi-tochondrial DNA) is a 16 569 - bp double - stranded, closed circular molecule, which encodes polypeptides participating in the oxidative phosphorylation and synthesis of ATP. However, not like the nuclear DNA, mtDNA is more susceptible to damage by exogenous mutagens and endogenous damage factors owing to many aspects, such as lack of protection by histones and effective mismatch repair (MMR) system, and the internal environment with high levels of free radicals and reactive oxygen species ( ROS) generated from oxygen in the organelle. So the injury severity and mutant rate of mtDNA were significantly higher than those of nuclear DNA ( around 10 times). The existing research data indicate that the biological features of tumors are not only decided by nuclear genetic materials, but also related with extranuclear mtDNA.The transcripts of mtDNA in tumors frequently increased, meanwhile ROS can influence mtDNA expression, which might decrease cell apoptosis and consequently be related with carcinogenesis. In addition, mtDNA copy number always happens to change, but there have not coincident conclusion about changes of mtDNA copy numbers in tumors. ROS can increase efficient damage to mtDNA; conversely accumulative mtDNA mutation could result in ROS generation. Radiotherapeutic killing effect on tumors is indirectly resulted from ionization of H2O molecule, therefore causing a great number of ROS, which damage macro-molecule and lead to tumor cell death. What on earth the relationship are therebetween the mtDNA transcripts and hypoxic resistance.ObjectiveIn this paper, we detected the transcriptional expression of mitochondrial genes C01, ND4, ND5, cyt - b and ATPase6, mtDNA copy number and variations of mitochondrial D -loop, 12S rRNA, ND4, tRNAphe and ATPase6 in gastric cancer and matched normal gastric mucosa, and further discussed the relationship between mtDNA subjected by damage and gastric carcinogenesis and their pathogenesis. In another study, we observed and evaluated changes of mtDNA transcripts under various time - phase hypoxic dispositions, and further discussed hypoxic influence on mtDNA transcriptional expression, and what roles mtRNA changes might play in hypoxic resistance to radioactivity.Materials and MethodsMaterialsForty - two matched gastric cancerous tissues and adjacent normal gastric mucosal tissues were obtained from the specimens of 42 patients with gastric carcinoma in the First Affiliated Hospital of China Medical University from 1999 to 2002. Another 20 cases of matched gastric cancerous were preserved by our department in low temperature freezer. All tissues were immediately frozen in liquid nitrogen after resection, stored at -80℃, and diagnosed according to WHO s histological classification. -Gastric cancer MGC803 cell line was preserved by our department.MethodsDetection of mutation and instability of mtDNA in gastric cancersVariations of mitochondrial D - loop, 12S rRNA, C01 and ND4 in 44 samples including 22 from gastric carcinoma tissue and 22 from adjacent normal tissues were detected by denaturing high performance liquid chromatography ( DH-PLC ) and direct DNA sequencing. Then laser capture microdissection technique ( LCM ) was used to separate the cancerous cells and dysplasia cells from the pa-tients with specific mutations. Allele - specific PCR (AS - PCR) , nest - PCR and polyacrylamide gel electrophoresis ( PAGE) were used to further evaluate this mutant property and quantitative difference of mutant type between cancerous and dysplasia cells. Finally, RNAdraw bio - soft was used to analyze the RNA secondary structure of mutant type.Detection of mtDNA copy number by PCR and PAGEHVt (hypervariable region) and HV2 of mitochondrial D -loop region from 20 cases of gastric cancer and 20 paracancerous tissues were amplified by PCR; meantime β - actin was served as a quantitative standard marker, followed by polyacrylamide gel electrophoresis ( PAGE) and silver staining, in which the difference of mtDNA copy number was compared between gastric cancers and paracancerous tissues.Qualification of mtDNA transcripts in gastric cancers and hypoxic MGC803 cell lineRT - PCR was applied to detect the transcripts of CO I , ND4, ND5, cyt-b and ATPase6 in matched gastric cancers and normal tissues, and varioustime phase hypoxic MGC803 cell line, followed by X - ray irradiation. Flow cy-tometry and colony forming test were conducted to evaluate the cell cycle phaseand survival fraction.Statistic analysisQuantitative data were expressed with mean ± standard deviation (SD). All data were disposed by SPSS 10. 0 statistical software to analysis the statistical differences between different groups via t - test, One - Way ANOVA and correlation coefficient. A value of P <0.05 was considered significant, and P <0.01 remarkably significant.ResultsmtDNA instabilities and mutations in gastric cancersFrequencies of mtDNA variations in D - loop, 12S rRNA, CO I , ND4 and ATPase6 were 18. 2% , 9. 1% , 13. 6% , 0. 0% and 0. 0% respectively. Impaired frequency of 12S rRNA alleles in intestine type of gastric cancers washigher than diffused gastric cancers, P <0. 05. np716 mutation of 12S rRNA was heteroplasmic and its quality in gastric cancer was higher than that in dys-plasia. RNA secondary structure analysis showed np652 G insertion was more disadvantageous to the stability of 12S rRNA RNA secondary structure, while 716 T - G transversion and other variations had only limited effects.Differences of mtDNA copy number in gastric cancers and normal tissuesAfter standardization withβ - actin, there was significant difference in HVt and HV2 copy number between gastric cancers and normal tissues, P <0. 01. That is, mtDNA copy number in gastric cancer and normal gastric tissues was different, the latter of which was low. mtDNA copy number was not associated with WHOs classification, Laurens classification or invasive depth, but with nuclear genome coded enzymes, such as AKP and cAMP-PDE, P<0.05.Transcriptional levels of mtDNA in gastric cancers and normal tissues Comparing to normal tissues, mitochondrial CO1 and ND4 transcripts in gastric cancers were higher (P <0. 01) , and higher in poorly differentiated gastric cancer than in well differentiated gastric cancers. Transcriptional levels of mtDNA were correlated negatively with histological differentiation of gastric cancers. According to Laurens classification, ND4 and CO1 expression levels in intestine type of gastric cancers were higher than those in diffused type, P <0. 05.mtDNA transcripts of MGC803 cells under hypoxia plus irradiation With the elongation of hypoxic time, CO1 transcripts showed remarkable decrease, and lowed to one fourth of that in pre - hypoxia after hypoxia for 24 h. ND4 transcripts elevated initially, increased to two times under hypoxia for 8h, then diminished to one second after hypoxia for 24h. Hypoxia leaded to blocked Gl phase and decreased S phase of MGC803 cell cycle, and induced hypoxic resistance to irradiation.ConclusionMost of variations exist both in gastric carcinomas and in normal tissues,...