Contrast Of MtDNA Variation In Esophageal Cancer Between Patients From High Risk Area And Normal Risk Area

Background There is a high risk for esophageal cancer in China, especially in north ofHenan province. People did a lot of work to detect why and how cancer happened on nuclear DNA, such as: the activation of oncogene, the inactivation of cancer suppressive gene. Recently , researchs have began to reach genetic substance outside nDNA, that is mtDNA.mtDNA is a kind of ringed double-chain DNA molecule that consists of 16569 bp. Its genome has two parts: control region and coding region. There is no intron in mtDNA. The replication origination and transcription promoter of mtDNA lie in the control region~D-Loop region, which controls the replication and transcription of mtDNA. The coding region codes 13 kinds of protein submits which are required in OXPHOS. It also codes 22 kinds of tRNA and 2 kinds of rRNA which are required to produce ATP. OXPHOS provides body more than 90% energy, which is conserved as ATP. Somatic mtDNA mutations have been found in cancers of head and neck, breast , lung, gastric, esophageal, hepatic, pancreatic, colorectal, bladder, prostate, ovarian and renal carcinoma, the control region—D-loop region is a hot mutation region. Mutations in this region may interrupt the mitochondrial function and play an important role in causing cancers.Two recent reports investigated the frequency of D-loop region mutation in esophagealcancer. In Japan, a place with normal risk of esophageal cancer, a study showed that D-loop region mutation rate in esophageal was only 5%(2/37) of specimens analyzed. But another study in Japan showed that HV-1 and HV-2 in D-loop region mutation rate in esophageal was34%(13/38).Objective One of objectives of our study is to find the mtDNA D-loop region variations inesophageal cancer. Many researches have argued that there is a close relationship between mtDNA variation and the origination of tumor. So we contrast the variations in esophageal cancer between patients from high risk area and normal risk area to look for new mechanism in the origination of esophageal cancer.Methods Extracting nDNA and mtDNA from 24 cases esophageal cancers Synthesizing apair of primer to amplify D-loop region by PCR(MF 15990 5'-CTTTAACTCCACCATTAGCACC-3' , MR583 5'-GCTTTGAGGAGGTAAGCTAC-3' ). PCR product was identified by 1.5% agarose gels electrophoresis and then was purified .The purified product was identified again by agarose gels and was recombined with T-vector. Then the recombination was transformed into the JM109. Using Amp fastness and blue/white color screening to select the positive colonies of transformed bacteria. Primer T7/SP6 was used to identify the positive clones to found whether the aim DNA was transformed into the germ or not. Identified positive clone was sent to be sequenced. Variations were found through contrasting the sequenced results and the sequence from GenBank by DNASIS soft. Differences were found between variations in esophageal cancer and variations in the adjacent normal esophageal tissues, and found between variations in esophageal cancer that come from high risk area and variations in esophageal cancer from normal risk area through rank sum test.Results1 There are 4 hot variations in the total 44 variations found in this study: 16223 site, C-T, 17 cases; 16362 site, T-C, 15 cases; 73 site, T-C, 15 cases; 75 site, inserting GC, 13 cases.2 Variations Only found in esophageal cancer tissues: 16182 site, inserting C; 16201 site, C-A; 16293 site, A-C; 16313 site, C-A; 16384 site, G-A; 16386 site, T-A; 16465 site, C-A; 16484 site, T-G; 16515 site, A-G; 16558 site, G-A; 36 site, inserting G; 52 site, insertingG; 82 site, inserting A; 132 site, C-T; 135 site, T-G; 178 site, inserting T.3 Variations Only found in esophageal cancer tissues from high risk area: 36 site, inserting G/A.4 Several cases which varied mostly: case 17,32 variations; case 19,35 variations; case 27, 25 variations; case 8, 24 variations. All these four cases are from esophageal cancer tissues and three of them are from the high risk area.5 Rank sum test was done on the amount of variations in tumors and in the adjacent normal esophageal tissues. T=68.5, which went beyond the T-value 39-66(P<0.05). If P-value of <0.05 was considered to be statistically significant. It can be said that mtDNA D-loop region variations have obviously difference between tumors and adjacent normal esophageal tissues6 Rank sum test was done on the amount of variations in esophageal cancer tissues from high risk area and in esophageal cancer tissues from normal risk area. T=56,which did not go beyond T-value 35-67 (P<0.05). If P-value of <0.05 was considered to be statistically significant. It can not be said that mtDNA D-loop region variations have obviously difference between tumors from high risk area and tumors from normal risk area.Conclusions1 MtDNA D-loop region varies a lot in esophageal cancer. These variations include polymorphism and mutations.2 MtDNA D-loop region variations have obviously difference between tumors and adjacent normal esophageal tissues, which shows variation is probably an accumulation process and it may play a role in the development of esophageal cancer.3 MtDNA D-loop region variations have no obviously difference between tumors from high risk area and tumors from normal risk area. But a/an G/A is inserted in site 36, which can only be found in tumors from high risk area. And three of those which varies mostly are all form high risk area. All these may show that there are peculiar variations in tumors from high risk area.