Study On Mitochondrial DNA Mutations And Alteration Of Copy Numbers In Breast Cancer

ObjectBreast cancer is one of the most common cancers of female in the world. Mutations in the mitochondrial DNA(mtDNA) have been observed in human neoplasia. The control region (D-loop)of mtDNA has tendency to accumulate mutations more rapidly than other regions of mtDNA. The first aim of this paper is to establish methods of detection the mutations of D-loop and the alteration of copy numbers of mtDNA by studying on the two cell lines MCF-7 and MDA-MB-231. The second is to determine the frequency and distribution of mutations in D-loop of mitochondrial genome by analysing tissues of breast cancer patients and blood of members of family breast cancer and to study the mechanism that genearate such mutations and impact of mtDNA mutations in tumor development. Furthermore, we detected alteration of copy numbers of these samples to exploit the relationship between D-loop region of mtDNA and generation of breast cancer and the relationship with susceptibility of genetic breast cancer.MethodsSection one: We cutured two breast cancer cell lines MCF-7 and MDA-MB-231 in vitro, then mtDNA was extracted from cells in logarithm increment phase. The segment of mtDNA encompassing the D-loop was amplified and ligated with T-vector then cloned in E. coli DH5a to sequcence and mutations were analysed. As the standard of real-time PCR, the T-vector including D-loop wasextraced from DH5a , and then absolute copy numbers of two cell lines were detected by real-time PCR amplification.Section two: We obtained 30 tissues of breast cancer ,30 whole blood samples from patients of breast cancer , 42 whole blood samples from family breast cancer and 30 whole blood samples from healthy blood donors as control . Total DNA of these samples were extracted with E. N. Z.A blood DNA kit. We designed three couples of primer with Oligo 6.2 software so that D-loop region were departed to three segments and then amplified with PCR. PCR products were selected with SSCP by electrophoresis. Then PCR products that occurrence mutations in the region of 128-599 of D-loop encompassing H-strand origin and D-310 region which was the hotspot of microsatellite instablity were sequenced directedtly. The frequence and distribution of mutions were analysed using DNASTAR software by comparing sequce result with Combridge sequce what was publicated. The changings of sequce were charged to polymorism or new polymorism and mutations.Section three: We used the T-vector linked D-loop of mtDNA that obtained from the first section as the standard, detected quantitively alteration of copy numbers of the mtDNA of the samples same as the second section with real-time PCR and analysed divergent of four groups.ResultsSection one: We sequenced the D-loop region of mtDNA of two cell lines and found nine mutations, respectively. The frequence ofmutations of the two cell lines is 0. 8%. These mutations including point and deletion/insertion mutations. In these mutations, 4 mutations of MCF-7 and 2 mutiations of MDA-MB 231 lie in original region of replication and transcription of H strand. Both of them present mutations in c-stretch(D-310). Moreover, a complex rearrangement was found in MDA-MB-231. Using real-time PCR, copy numbers of mtDNA of two cell lines that we detected are 4.84 Xl015and 5. 63X1015 copy/ugDNA.Section two: We found mutations in 15 of the 30 (50%) breast cancer tissues and 13 of the 30(43.3%) whole blood of breast caner patients which we screened with PCR-SSCP. Both of them are statistic significantly higher than control (6%) (P<0. 05) . There are 66 mutations in 12 tissue samples which were found in the region of 128-499 of D-loop: 51 are point mutations, 23 of them are T C, 15 are A G;5 are insert mutations;10 are deletion mutations. 23 mutantions that we found in all of 12 samples occurranced in Ostretch. 52 of these mutations are polymorism that have been found, other 14 are new polymorism and mutations. There are the same mutations of 263 A G and 489 T C lie in all of 12 samples. Mutations occurance in D310 is differtent in every tis...