Mutations Of MtDNA And Expressions Of Mt-mRNA, VEGF, BRCA₁ And PTEN In Invasive Ductual Breast Carcinoma

Breast carcinoma is a primary and malignant to the female. It is known that gene mutation and expression are the foundation of exasperated cell and is nearly correlative to tumour occurring. Mitochondrial deoxyribonucleic acid (mtDNA) is the only germ out of nuclear deoxyribonucleic acid (nDNA) and it could easily become attacked target by carcinogen, because of lacking of histone protecting and effect repairing system. It is known that mtDNA is related to many diseases and tumours. The control region for replication (D-loop region) is very important in the noncoding region of mtDNA, which participate in and intermediate mtDNA replication and transcription. However, the difference of the region could be involved mtDNA to the changes of replication and transcription. The cytochrome b gene (cyt-b gene) of mtDNA could encode one subunit of the complexâ…¢in oxidative phosphorylate respiration chain, evolved to the speed moderate and stabilation. In recent years, lots of special mtDNA mutations in gene stabilize region about disease were reported, which offered reliable thereunder for diseases diagnose and research. Vascular endothelial growth factor (VEGF), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and breast and ovarian cancer suppressor gene I (BRCA₁) play a crucial role in the process of growth and metastasis of breast carcinoma; these have turned into the research focus in the tumour. But the relationship between VEGF and breast carcinoma has not repoted.Objeetive: 1. To detect the somatic mutations in mtDNA HV2 region of D-loop region and to investigate the role of mtDNA mutation in the tumorigenesis of the breast carcinoma. 2. To detect the cyt-b gene alterations in the carcinoma tissue,the normal paracarcinoma tissue and the blood sample of the same breast carcinoma, and to search special mutation spots. 3. To detect the copy numbers of mtDNA in breast carcinoma. 4. To detect the expression of the mtDNA cyt-b and VEGF in breast carcinoma; and investigate the role and relationship of the factors. in breast carcinoma; 5. To detect the protein expression of the VEGF, PTEN and BRCA₁ and the microvessel density (MVD), and to investigate the role and relationship of the factors in breast carcinoma.Methods: 1. The region of coding mtDNA cyt-b gene of the carcinoma tissue, the normal paracarcinoma tissue and the blood sample in 20 breast carcinoma patients were analyzed by PCR and PCR-single stranded conformation polymorism (PCR-SSCP), and there being exceptional band cases did DNA sequencing. 2. The hypervariable regionâ…¡(HV₂) of nocoding D-loop in mtDNA was analyzed by PCR and gene sequencing in the positive cases of above experimentation. 3. The hypervariable regionâ… (HV₁) andâ…¡(HV₂) of the mitochondrial D-loop region from 20 cases of breast carcinoma tissues and 20 cases of normal paracarcinoma tissues were amplified by PCR; and meantimeβ-actin, one of the nuclear genes, was served as a quantitative standard marker, followed by polyacrylamide gel electrophoresis (PAGE) and silver staining, in which the difference of mtDNA copy number was compared between the breast carcinoma and normal paracarcinoma tissues. 4. The mtDNA cyt-b and VEGF mRNA from 19 cases of breast carcinoma tissues and 19 cases of normal paracarcinoma tissues were amplified by reverse transcription polymerase chain reaction (RT-PCR); and meantimeβ-actin was served as a quantitative standard marker, and the differences of mRNA expression were compared between breast carcinoma and normal paracarcinoma tissues. 5. The expression levels of VEGF and PTEN and BRCA₁ protein were assessed in 42 cases of breast carcinoma tissues and 30 cases paracarcinoma tissues with S-P immunohistochemical method, and the differences were compared between breast carcinoma and normal paracarcinoma tissues.Results: 1. The 7 exceptional bands were found from 20 breast carcinoma in all of the samples by PCR-SSCP, and the sequencing results indicated that there were 6 high mutation rate spots as follow: nt15326A→G(91%, 20/21); nt14766C→T(81%, 17/21); nt15301G→A(52%, 11/21); nt15043G→A(48%, 10/21); nt14783T→C(43%, 9/21); nt15402C→T(35%, 7/21). 2. The 31 new HV₂ mutations were found in 7 sequencing specimens, the 3 of them were mitochondrial microsatellite instability (MSI); the 7 of them were frameshift mutations; others were point mutation; and the 7 of them located in the region of mtDNA replication and transcription. 3. There copy number of mtDNA HV₁ and HV₂ (standardized withβ-actin) in the breast carcinoma tissue was lower than the normal paracarcinoma tissue (p＜0.05 in HV₁ and p＜0.01 in HV₂). 4. The mtDNA cyt-b expression and VEGF mRNA expression were stronger in the breast carcinoma tissue than in the normal paracarcinoma tissues (p＜0.01), and the mtDNA cyt-b expression was positively correlated with VEGF mRNA expression (p＜0.01). 5. The expression of VEGF protein was higher in the breast carcinoma tissue than in the normal paracarcinoma tissue (p＜0.01); the expression of VEGF protein was positively correlated with VEGF mRNA expression (p＜0.01), and the MVD were positively correlated with the VEGF protein expression (p＜0.05). 6. The expression of PTEN and BRCA₁ was lower in the breast carcinoma tissue than in the normal paracarcinoma tissue (p＜0.01). 7. The expressions of the PTEN and the BECA₁ were negatively correlated with the expression of the VEGF pretein (p＜0.05).Conclusions: 1. The mtDNA HV2 region is a highly polymorphic and mutatble region, that could be some relations in the breast carcinoma development. 2. The 6 high mutation spots of mtDNA cyt-b are detected in the breast carcinoma tissue and the nt15326C→T and nt14766C→T are the hottest spots in breast carcinoma. 3. Less mtDNA copy number in the breast carcinoma tissue could be treated'as clinical diagnosing guideline of the breast carcinoma. 4. The high expressions of mtDNA cyt-b and VEFG mRNA in the breast carcinoma tissue indicate that they could accerate the breast carcinoma development by energy providing in the tumour cell. 5. The relationship of VEGF protein expression with PTEN and BRCA₁ in the breast carcinoma tissue could be the impersonal guideline of prognosis of the breast carcinoma, and VEGF expression and MVD could be used as a target of vascular inhibiter.