| Objective:Mitochondria can produce, store and supply energy. Mitochondrial DNA is independent of nuclear DNA. The recognition of the relationship between the mutation of mitochondrial DNA and probable diseases has dropped far behind the study on the nuclear DNA. In recent years, the instability, gene mutation and abnormal expression of mitochondrial genome has been detected in many kinds of malignant cancer tissues such as gastric cancer,colon cancer, and liver cancer, etc. In this report, the region of mtDNA D-loop was amplified by PCR and then sequenced with samples from cancer tissues and corresponding normal mucous membrane of 20 gastric cancer patients. Reactive oxygen species(ROS) and cell cycle were detected with flow cytometry. The samples were divided into mutation group and control group according to the mutations. We compared the level of ROS and cell cycle in two groups to clarify the influence of mutations of D-loop on ROS and cell cycle. Furthermore, we studied the r mismatch repair gene to invastigate the interaction between the mutations of mtDNA and nuclear DNA. Our purpose was toinvestigate the influence of these factors on cell carcinogenesis and progression of gastric cancer.Materials and Methods1. Twenty surgical samples of gastric cancer were selected from hospitalized patients in the Department of Ggastrointestinal Surgery of The First Affiliated Hospital of Nanjing Medical University from May to August, 2003. The D-loop region was amplified by PCR and sequenced in 20 specimens from gastric cancer cells and adjacent normal tissues.2. Some tissues were cleaned of blood remnant with PBS and prepared singular cell suspension at once.The level of ROS and cell cycle were detected by flow cytometry. According to the sequence results, the gastric cancer tissues were divided into mutation group and control group, and the level of ROS, apoptosis and hyperproliferation in the two groups were compared each other.3. Mitochondrial DNA microsatellite instability in tumor tissues was analysesed by PCR-SSCP, so was nuclear DNA BAT26 microsatellite instability.Results:1. Totally 206 single nucleotide polymorphisms(SNPs) were obtained ascompared with the sequence of mtDNA D-loop from Genbank, among them 197 were known and 9 SNPs from 5 patients were first reported. In the cancerous tissue of 7 patients, 18 gene mutations were found, among which 4 were microsatellite instabilities. Thus the mutation rate of mtDNA D-loop in the specimens of gastric cancer is 35%.2. The level of ROS in mutation group and control group were 156.3 ± 9.7(MFI) and 117.3 ± 10.4(MFI), respectively(P<0.01).The rate of apoptosis in mutation group was 12.5 ± 1.5 %, and the rate of apoptosis in control group was 8.6 ± 0.1 % (P<0.05). In cell cycle,DNA is synthesized in synthesis(S) phase. As a result, the percentage of cells in synthesis phase can reflect proliferation. The percentage of cells in synthesis phase in mutation group (22.3 ± 2.4%) was higher than that in control group( 16.4±1.5%) (P<0.01), which indicating the rate of proliferation in mutation group was also higher than that in control group.3. Six(30%) mtMSI were found, among them five were in D-loop region and one in coding region. One(5%) BAT26 microsatellite instability was detected without mtMSI.Conclusion:1. The mtDNA of D-loop region is highly polymorphoric and the mutation rate is relatively higher in patients with gastric cancer.2. The mutation leads to the dysfunction of resparatory and the increase of reactive oxygen species in gastric cancer cells.According to our results the content of ROS in mutation group is obviously higher than that in control, coincident with the phenomenon that the apoptosis in the former is obviously higher than that in the latter. ROS also play some roles in the obvious elevation of proliferation in mutation group, suggesting that as a primary factor ,the mutation of D-loop perhaps take part in the process of gastric cancer genesis and progression through the effect of the increased ROS— elevat...
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