Study Of Relationship Between Mitochondrial DNA And Carcinogesis, Progression,Cell Apoptosis In Esophageal Squamous Cell Carcinoma

Esophageal carcinoma is one of the most frequently diagnosed malignant tumors in the world.China is one of the high-risk areas of esophageal carcinoma,especially in Linzhou City(previously Linxian County),where esophageal carcinoma incidence and mortality rate are among the highest in the world.Therefore,many studies have been focused on the mechanism of esophageal carcinoma,early diagnosis and targeted therapy.Mitochondrial is one of the important organs in eukaryotic cells.Oxidative phosphorylation and energy conversion are mainly performed in the mitochondrial. Mitochondrial DNA(mtDNA) is a special extranuclear genetic material.Mt-DNA is divided into non-coding region(D-loop) and coding region.Compared with nuclear DNA,mt-DNA has relatively small molecular weight and lacks of the histone protection of the nuclear genome,i.e.it is bare.The mtDNA also has weak repair systems and is directly exposed to hyperoxia during oxidative phosphorylation,which is a major factor for the damage.Therefore,mtDNA is easier to be affected by oxidative damage and attacked by carcinogens and its degree of injury and mutation rate is markedly higher compared to the nuclear DNA.Carcinoma development is characterized by a complex series of phenomena occurring at the DNA level.Because carcinogenesis is resulted from the aberrant interplay of oncogenes and suppressor genes,it remains to be answered why and how mtDNA is mutated in tumor.The available data suggested that the tumor biological features not only depend on the intranuclear genetic material but also on the characteristics of the tumor extranuclear DNA.Tumorigenesis might be related to the fact that mtDNA is prone to damage and not easily repaired.There are now more than 50 disease-causing mtDNA mutations and hundreds of mtDNA rearrangement known. Over the past decade,many somatic mtDNA mutations have been identified in various tumor tissues,including bladder carcinomas,head and neck carcinomas,lung carcinomas,pancreatic carcinoma,hepatocellular carcinomas,colorectal carcinomas, thyroid carcinomas,breast carcinomas,prostate carcinoma,and gastric carcinomas. These findings have suggested that mtDNA mutations may be involved in the initiation of carcinogenesis.Changes in mitochondrial DNA have been reported in carcinoma cells.Since little information exists regarding the relationship between mt DNA and the occurrence,development of esophageal carcinoma(especially esophageal squamous cell carcinoma,ESCC) and apoptosis.The purpose of the present study is to further clarify this relationship between mtDNA and ESCC and provide an effective methods for the inhibition of occurrence,development of ESCC and a theoretical basis for the targeting treatment of ESCC.After mtDNA and nuclear DNA were extracted simultaneously from the same tissues of the 62 cases of ESCC,31 cases of atypical hyperplasia tissues of tumor-adjacent and 62 cases of normal esophageal mucosa using agent kit,their mtDNA copies were tested respectively using semi-quantitative PCR and real-time fluorescence quantitative PCR.The PCR products of ND1 gene of mtDNA respectively were purified and sequenced to understand the relationship between ND1 gene mutations and the occurrence,development of ESCC.Theρ°cells of the human esophageal carcinoma cells EC9706 and TE-13 were first successfully established,then the relationship between mtDNA copies and apoptosis were investigated using semi-quantitative PCR,real-time fluorescence quantitative PCR,TUNEL staining and flow cytometry.This study is divided into the following three parts.PartⅠChange in the mtDNA copies and its significance in ESCCMethods1.mtDNA coding region(3490bp-3892bp,about 403bp) the 62 cases of ESCC, 31 cases of atypical hyperplasia tissues of tumor-adjacent and 62 cases of normal esophageal mucosa were amplified respectively and their copies were determined using semi-quantitative PCR and real-time fluorescence quantitative PCR.PCR products were validated by agarose gel electrophoresis.β-actin gene was also amplified as control.2.Statistical treatment:Statistics analysis was performed by SPSS 13.0 software, adopt chi-square test,t test and ANOVA,test standardα=0.05.Results1.The relative contents of mtDNA extracted from the above tissues were 1.604±0.195,1.533±0.147,1.401±0.185,respectively.The results suggested that the relative contents of mtDNA in ESCC tissue were higher than those in paraneoplastic and normal esophageal tissue(P＜0.05).There were significant differences (F=19.536,p＜0.05) in mtDNA among the three tissues.2.The detection rate of mtDNA was 100%in the above three tissues.The average mtDNA copies(Ct values) detected by the real-time fluorescence quantitative PCR were 25.394±1.380,26.068±1.792,26.835±1.949,respectively in the above three tissues.There were significant differences(F=11.020,p＜0.05) in mtDNA copies among the three tissues.PartⅡStudies of ND1 gene mutations of mtDNA encoding region ND1 gene and the correlation with mtDNA copies in ESCCMethods 1.ND1 genes of mtDNA coding region in the 31 cases of ESCC,31 cases of atypical hyperplasia tissues of tumor-adjacent and 31 cases of normal esophageal mucosa were amplified respectively using PCR.2.The PCR products of ND1 genes were recovered,purified and sequenced respectively.3.Statistical treatment:Statistics analysis was performed by SPSS 13.0 software, adopt chi-square test,t test and ANOVA,test standardα=0.05.Results1.The identical mutations in the same position(C3971T,T3553A) were found in 9 cases out of 31 cases of atypical hyperplasia tissues of tumor-adjacent(36.67%).No mutations of mtDNA ND1 gene were found in the 31 cases of normal esophageal mucosa.2.The difference of the mtDNA copies between ND1 gene mutant region and non-mutant region was statistically significant(P＜0.05).The average mtDNA copies(Ct values) detected by the real-time fluorescence quantitative PCR were 25.558±1.0789,26.324±1.897,26.932±0.530,respectively in the above three tissues. Mutations were found both in 9 cases of ESCC and atypical hyperplasia tissues of tumor-adjacent.Their Ct values were 24.774±1.129,23.921±1.0574 and have significant differences(F=5.184,14.861,P＜0.05).PartⅢStudies of the relationship between mtDNA and apoptosis in ESCC ceU Unes of EC9706 and TE-13Methods1.Cells deficient in mtDNA(ρ°cells) were acquired from ESCC cell lines EC9706 and TE-13 mutagenized by EB.2.MtDNA copies of the two cell lines were detected at different time using the real-time fluorescence quantitative PCR after treated by EB.PCR products were validated by agarose gel electrophoresis. 3.ND1 genes of mtDNA coding region in ESCC cell lines EC9706 and TE-13 were amplified respectively using PCR.The PCR products of ND1 genes were recovered,purified and sequenced respectively.4.Apoptosis of ESCC cell lines EC9706 and TE-13 were analyzed using TUNEL staining and flow cytometry at different time after treated by EB.5.Statistical treatment:Statistics analysis was performed by SPSS 13.0 software, adopt chi-square test,t test and ANOVA,test standardα=0.05.Results1.Theρ°cells of ESCC cell lines EC9706 and TE-13 were first successfully established.The results identified by the real-time fluorescence quantitative PCR indicated that mtDNA copies decreased progressively with the increasing in times of cell division in the presence of EB and mtDNA was disappear until 12 days.2.Mutation C3971T in mtDNA ND1 gene was found both in ESCC cell lines EC9706 and TE-13.3.mtDNA copies ofρ°cell line from ESCC EC9706 and TE-13 were tested on the 4th,8th,12th day after the cells were treated with EB.Normal cells were also performed as the controls.The results showed that the mtDNA copies in the EB-treated groups decreased gradually from the 4th day to the 12th day compared with the controls'.mtDNA copies was zero on the 12th day.4.Apoptosis analysis firstly were performed during the culture ofρ°cells of EC9706 and TE-13 respectively after the cells were treated with EB on the 4th,8th and 12th day.The results detected by flow cytometry indicated that apoptosis was increased gradually from the 4th day to 12th day.Apoptotic rates(%) were 16.42±0.70,59.80±0.49,68.80±0.21 in the cells TE-13 and 2.78±1.04,11.68±1.85,26.62±1.06 in the cells EC9706.The results deteded by tunel were consistent with apoptosis was increased gradually from the 4th day to 12th day.Conclusions1.It is timesaving and economical to extract mtDNA and Ndna simultaneously from the same tissues.Studies of the correlation between mtDNA and nDNA using this method are more persuasive.2.mtDNAs of different tissues in tumorigenesis and progression of ESCC were determined using semi-quantitative PCR.We found that the mtDNA copies were increased in the normal tissues,atypical hyperplasia tissues of tumor-adjacent and ESCC.These data suggested that increase of mtDNA copies are related to the occurrence and development of ESCC.3.We also found that mtDNA copies determined by the real-time fluorescence quantitative PCR tended to increase gradually in the the normal tissues,atypical hyperplasia tissues of tumor-adjacent and ESCC,which suggested that increase of mtDNA copies are closely correlated to the occurrence and development of ESCC.4.Sequencing analysis of mtDNA ND1 genes(C3971T,T 3553A) in various lesions of esophageal tissues indicated that 29.03%of ND1 genes in the atypical hyperplasia tissues of tumor-adjacent and ESCC were found to be mutagenized.No mutations were found in the nomal tissues.These results suggested that mtDNA mutations may be play certain role in the occurrence and development of ESCC.This is further confirmed that Mutations of C3971T were also found in the mtDNA ND1 genes of EC9706 and TE-13 cells through sequencing.5.MtDNA copies of the mutant group are obviously higher than that of the control group and have significant differences(P＜0.05).This suggested that muDNA mutations are closely related to mtDNA copies.6.Establishment of theρ°cells of ESCC cell lines EC9706 and TE-13 offers new tools for research on the relationship between mitochondrial DNA and esophageal carcinoma.7.Apoptotic rates of EC9706 and TE-13 cells were both increased gradually with the decrease of mtDNA copies in the two cell lines.The results suggested that mtDNA may patticipate in the inducement of apoptosis.This suggested that mtDNA lesions induced selectively could lead to obvious decrease and further induce cell apoptosis.This is wished to become a new target for biotherapy of esophageal carcinoma. Innovations1.The method of extracting mtDNA and nDNA simultaneously from the same tissues is timesaving and economical.Studies of the correlation between mtDNA and nDNA using this method in the research of carcinoma and related diseases are more persuasive.2.Copies of mtDNA and ND1 gene in different tissues and stages of ESCC were determined using semi-quantitative PCR and the real-time fluorescence quantitative PCR.3.ND1 genes of mtDNA in the normal esophageal mucosa,atypical hyperplasia tissues of tumor-adjacent and ESCC were amplified,purified and sequenced.Point mutations(C3971T,T3553A) were found in ND1 gene in atypical hyperplasia tissues of tumor-adjacent and ESCC.4.Cell linesρ°of ESCC EC9706 and TE-13 were firstly successfully established. It offers new tools for research on the relationship between mitochondrial and esophageal carcinoma.5.The correlation between changes of mtDNA copies and apoptosis in ESCC cell lines EC9706 and TE-13.ND1 genes of mtDNA in the two cell lines were sequenced and point mutation(C3971T)was found in their ND1 genes.6.Copies of mtDNA in the two cells was gradually reduced.Up to 12d,the copy .was zero.And the apoptosis cells were gradually increase.