Detection And Analysis Of Mitochondrial DNA Mutation In Colorectal Carcinoma

Background and ObjectiveThe morbidity and mortality of colorectal cancinoma(CRC), one of mostcommon malignant gastrointestinal tumors, were gradually increasing from the 1990s of twentieth century and now are similar all over the world. The mobidity of CRC remains the fouth in that of malignant neoplasm in the world , and the same in our country . The age of CRC patients becomes yonger than before. The incidence of CRC is approximately from 24 to 32 cases per one hundred thousand in the population of older than 35 years. CRC has been up to the fourth leading cause of death in cancer.With the development of molecular biology, genetics and biotechnology, the CRC researches concerning its basis , clinics and preventions have been expanded . However, the mechanism of CRC occurrence, reoccurrence and metastasis remains unclear . The mass screening in high risk population (more than 40 years old , positive family history ,or patients with ployps ) could help to find CRC in the early stage . However ,with a view of limited colonoscopy application, little effects caused by small CRC lesions , few clinical manifestations, and poor specificity of CEA detection , there are still lack of simple and effective methods for finding out CRC in the early stage nowadays . It is very important of the identification of tumor markers and special or associated antigens to diagnose CRC in eary stage treatpatients by early surgery , and increase the survival rate of five years.Finding suggests biological specificity of tumor is not only associated with nuclear DNA but also have much importantce with the mutations of mitochondrial DAN(mtDNA), because mtDNA is lack of protection of the histone and effective injury-repair mechanism in contrasted to nuclear DNA, so mtDNA is easily attacked by carcinogenetic matter and become the target of carcinogenesis. The research on mtDNA such as the mutation of mtDNA and mitochondrial genome instability(mtGI) in solid tumors have been reported recently, also detected in leukocyte of patients with cancer ,but the relationship of the mutation of mtDNA and carcinogenesis is still unclear.This research will analyze the character and rule of mutation of mtDNA in colorectal carcinoma cell lines and tissues , go further study the relationship between the mutation of mtDNA and colorectal carcinogenesis and investigate the mechanism and role of the mutation of mtDNA in colorectal carcinogenesis , expect to find a new marker for detecting colorectal carcinoma in early stage, so that we can treat patients with colorectal carcinoma by early stage and improve the rate of five years. Methods:1. to amplify the D-loop region gene of mtDNA in four colorectal carcinoma cell lines such as SW620 ,SW480, LOVO and HT29 and one normal colorectal cell line and succeeded , then purify the PCR production and sequence the gene of D-loop region of mtDNA in ABI377 seqencer made in America, lastly contrasted with the mtDNA genebank data to analyze the mutations of D-loop region gene of mtDNA in four colorectal carcinoma cell lines.2. Extract the mtDNA of colorectal carcinoma tissue and normal colorectal epithelial tissue of 40 patients with colorectal carcinomas, then design four pairsof primers to amplify the D-loop region gene of mtDNA in every colorectal carcinoma tissue and normal colorectal epithelial tissue. Lastly silver stained single strand conformation polymorphism (SSCP) was used to detect point mutation in the D-loop region gene of mtDNA in every colorectal carcinoma tissue by abnormal running gel.3. Extract the mtDNA of leucocyte of peripheral blood in 20 patients from 20 colorectal carcinoma and 6 nomal person, then design one pair PCR primer according to the whole gene of D-loop region to amplify the D-loop region gene of mitochondrial DNA of peripheral blood leucocyte , then directly sequencing the gene of D-loop region of mtDNA and analysis the mutations. Results:1. Point mutations were foud in four colorectal carcinoma cell lines and one normal colorectal cell line .The C to T mutation at np72, A to G mutation at np73, C to T mutation at npl6298, T to C mutation at npl6519 were found in four colorectal carcinoma cell lines and one normal colorectal cell line, these mutations was associated with polymorphism. One insertion sequence was found between np64 and np65 in LOVO cell line, The T to C mutation at npl6311 were found in SW480, LOVO and SW480 cell lines, the C to T mutation at npll4, np498 and npl6234 were found in SW480 ,SW620 and HT29, npl6224 and npl6311 was found in SW480 and LOVO, the same mutations found in different colorectal carcinoma cell lines were associated with typical point mutation.2. 7 abnormal electrophoresis strips can be found in 40 colorectal carcinoma tissues with corresponding normal colorectal epithelial tissues using PCR-SSCP technique, the frequency of abornomal electrophoresis strips can be found in four primers are 1, 4, 3, 1 and 2 point mutations in the same colorectalcarcinoma tissues can be found in PCR products produced by primer 2 and 3.3. Point mutations were foud in the D-loop region gene of Mitochondrial DNA of peripheral blood leucocyte in 20 colorectal carcinoma patients and 6 normal person. The C to T mutation at np72, A to G mutation at np73, T to C mutation at npl6519 were found in the D-loop region gene of mitochondrial DNA of peripheral blood leucocyte in 20 colorectal carcinomas and 6 normal person , these mutations was associated with polymorphism. The C to T mutation at np498 , npl6234, npl6260 , can be found in patients of colorectal carcinoma ,while not found in normal person .Other mutations are spontaneously occurred.Conclusions:1. Mutations found in colorectal carcinoma cell lines and normal colorectal epithelial cell are associated with polymorphism . Because DNA sequence of mtDNA is detected from westerners by Sanger in 1981, polymorphism found in colorectal carcinoma cell lines and one normal colorectal epithelial cell detected from easterners may be associated with different ethnic group and race. The same mutations found in different colorectal carcinoma cell lines are thought to be characteristic change and the characteristic change may be associated with colorectal carcinoma susceptibility, but relationship between characteristic change and carcinogenesis needs further study.2. A certain degree point mutations can be found in the D-loop region of mtDNA incolorectal carcinoma tissues, occurrence of mutation may be associated with susceptibility and grade malignance ,but whether the mutations have carcinogenicity needs further study.3. Similar mutations can be found in the D-loop region of mtDNA in peripheral blood leucocyte of patients with colorectal carcinoma and colorectal carcinomacell lines, but further study needs to identify the marker of colorectal carcinoma...