The Ratio of ssDNA to dsDNA in Circulating Cell-Free DNA Extract is a Stable Indicator for Diagnosis of Gastric Cancer.

Due to the different mechanisms of cell-free DNA production, the single-stranded DNA to double-stranded DNA ratio in blood maybe different between healthy individuals and gastric cancer (GC) patients. We aimed to explore the potential application of this ratio in GC diagnosis. The plasma cell-free DNA extracts from 118 healthy individuals and 106 GC patients were prepared. The levels of single-stranded DNA or double-stranded DNA in plasma, and the single-stranded DNA to double-stranded DNA ratio on the diagnostic efficiency for GC were assessed with ROC curve. The relationships between this ratio and the clinical characteristics of GC patients were analyzed. The ratios in 63 GC patients before and after surgery were compared. In healthy individuals, the single-stranded DNA to double-stranded DNA ratio was not affected by factors including age, gender and BMI, and subjected to normal distribution (P = 0.1090). GC patients had a lower value of this ratio than healthy individuals (P < 0.0001). Considering this ratio as a GC diagnostic indicator, the area under ROC curve (AUC) was 0.923[95% confidence interval (CI):0.880-0.955]. This ratio in unresectable GC was obviously lower than that in resectable GC (P = 0.0045). There was a rank correlation between this ratio and GC TNM staging (rho = -0.266, P = 0.0058), but it had no correlation with tumor size (r = 0.14, P = 0.145). Additionally, this ratio was not affected by hemolysis and repeated freeze-thaw of blood samples, and was significantly elevated after surgery(P < 0.0001). The single-stranded DNA to double-stranded DNA ratio in plasma is a stable non-invasive indicator for GC diagnosis.

Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells.

To investigate the role of ROS in the helicobacter pylori (Hp) induced mtDNA mutations, AGS cells were treated by extracts of Hp11638 or Hp11638M. The ROS levels, cytochrome C reductions, and intracellular ATP levels were measured. The coding region and the D-Loop region were amplified and sequenced. Results showed the ROS levels, cytochrome C reduction and mtDNA mutations were markedly increased and cell viability decreased after treatment with both Hp extracts, and 616 mutations were detected in D-Loop region and 3 heteroplasmic point mutations in the Cytb gene. No mutations were found in the coding region. The mutation rates of mtDNA D-Loop region were positively correlated with the ROS levels and negatively to the ATP levels.

[Mitochondrial DNA mutations in gastric endothelial cells induced by extract of helicobacter pylori in vitro].

OBJECTIVE: To investigate the relationship between the helicobacter pylori (HP) infection and the genetic instability of mitochondrial DNA (mtDNA) in human gastric adenocarcinoma epithelial cells (AGS). METHODS: After treated with extracts of HP11638 (CagA+, VacA+) or Hp11638 mutant strain (CagA+, VacA-), AGS cells were collected, and mitochondrial DNA was extracted and Cox-I, Cox-II, Cox-III, ATPase6, ATPase8 and Cytb genes and the D-Loop region were amplified by PCR and then sequenced. RESULTS: The mutation rates of the mtDNA in AGS cells were correlated with the extracts of the two HP strains in a concentration- and time-dependent manner. But the mtDNA mutation rate in AGS cells treated with the HP11638 extract was higher than that treated with the Hp11638 mutant extract. Total of 616 mutations in D-Loop region were detected, including 489 point mutations, 81 insertions and 46 deletions. Among them, 70.9% (437/616) belonged to GC to AT and AT to GC transition. Seventeen out of 20 (85%) AGS cells treated with extract of HP had mutations in 303PolyC, 16184PolyC and 514CA regions of mtDNA D-Loop. No mutation was detected in Cox-I, Cox-II, Cox-III, ATPase6 and ATPase8 genes, three point mutations were found in the Cytb gene. CONCLUSION: HP can cause the accumulation of mutations in mtDNA, in particular, in the D-Loop region, and the VacA participated in the process.