MtDNA Mutation And Apoptosis Gene Expression In Primary Tumor And Plasma Of Gynecology Oncology Patients

Study objectives : By investigating the mutation rate, mutation categories and mutation hot points of mtDNA in tumor tissue as well as plasma in patients with solid tumor, and by investigating the gene expression of bcl-2,bax, and caspase, we tried to find out the possible role of somatic mutation of mtDNA in the process of apoptosis, tumorigenesis and tumor development. It might be of great significance in elucidating the molecular mechanism of tumorigenesis and in finding new and valuable tumor marker. So it might provide a new approach in tumor diagnosis, treatment and prognosis.Methods: Total 160 samples of tumors, their proximal normal tissues, peripherial blood monocytes(PBMC), and plasma of 32 gynecological malignant tumor patients as well as 8 gynecological benign tumor patients were taken. The malignants were 5 squarmas cervical carcinomas, 10 endometrium carcinomas and 17 epithelial ovarian cancers (EOC). Clinical stage : 19 cases were in early(â…  , â…¡) stage and 13 were in late stage. Histological grade: 24 cases were in grade 1-2 and 8 were in grade 3. The benign tumors were 4 ovarian epithelial tumors and 4 uterus myoma . High molecular weight genomic DNA isolation was performed by using standard phenol and chloroform extraction. PCR-SSCP was done to examine mtDNA mutation. RNA isolation and RT-PCR was done to examine bcl-2, bax, and caspase gene expression. Results:1. The rate of mtDNA mutation: We found out 32 mtDNA mutations which were distributed in 22 cases of~malignants, and 18 mtDNA polymorphisms. The mtDNA mutation rate and polymorphism rate were 68.8% ( 22/32 ) and 56.3% ( 18/32 ) respectively. There were 4 mtDNA mutation in 3 cases of benign patients , and 6 polymorphism in 4 cases of benigns. The mtDNA mutation rate and polymorphism rate were 37.5%( 3/8 ) and 50% ( 4/8 ) respectively. The difference between malignants and benigns was significant ( p<0. 05 ) .In different malignant tumors, the mtDNA mutation and polymorphism rates were 80% (4/5) and 60% (3/5) in cervical cancer, 60% (6/10) and 40% (4/10) in endometrium carcinoma, 70.6% (12/17) and 64.7% (11/17) in ovarian cancer.2. The hot point of mtDNA mutation: In total 32 mutation of malignants , 5 (15.6%) were locating in ND1, 7 (21.9%) in ND4, 3 (9.3%) in ND5, 11 (34. 3%) in Coll, and 16 (50%) in cytb gene region. In 4 mutations of benigns, 1 (25%)locating in ND4, 3 (75%)in cytb. The results indicated that cytb gene region was the hot point of mtDNA mutation.3.Multigene mutation of mtDNA: Only 8 had one gene mutation in total 22 cases of mtDNA mutaion, including 1 case in ND1 , 3 cases in cytb, 4 cases in Co II gene region. The mojarity (63.6%) of patients involved in multigene mutation. 9 cases had 2 gene mutation (1 in ND5+cytb gene, 1 in NDl+cytb, 2 in ND4+cytb, and -in 5 in Coll +cytb).l case had 3 gene mutation( ND4+ND5+cytb),and 3 cases had 4 gene mutation(1 in NDl+ND4+ND5+cytb, 2 in NDl+ND4+CoII+cytb).4. mtDNA mutation of plasma: The plasma of patients which had mtDNA mutation in tumor tissues were taken for PCR-SSCP to analysis mtDNA mutation. PBMC were used as self-control. 20/22(90%) of cases had PCR amplification successfully , and 9 cases (45%) had positive results which were corresponing with tumor tissues.5. The relationship of mtDNA mutation and apoptosis gene expression. In total 22 cases of mtDNA mutation, 20 cases (90. 9%) had bcl-2 co-expression. But bcl-2 expression rate was 50% in the mtDNA non-mutation group, there was significant difference between them ( P<0. 05 ) . Bax and caspase gene expression in the mtDNA mutation group were 27.3% and 13.6% respectively. They were much lower than that of mtDNA non-mutation group (all P<0. 01), indicating that mtDNA mutation had a positive relationship with bcl-2 gene expression, while it had a negative relationship with expression of Bax and caspase.Conclusions: There are high frequent mtDNA coding area mutation in gynecological malignant tumors. The total mutation rate was 68.8%. In different tumors the mutation rates are 80% (cervical cancer), 60% (endometrium cancer), and 70. 6%(ovarian cancer) respectively. Indicating that mtDNA coding region mutation is closely related to gynecological malignants development and progression. It may be used as an important molecular genetic alteration in mechanisms of tumorigenesis. The hot point of mtDNA mutation is in cytb gene region . Cytb gene mutation may has significant influence on oxidative phosphorylation of mitochondria. The characteristic of mtDNA mutation is multigene and multisites mutation. The mojarity (63.6%) of patients involved in 2 more gene mutation . The mutation rate of benign tumors is much lower than that of malignant tumors.It reveals that there is different molecular mechanisms between them.There is no significant difference among tumor type, clinical stage , and histologic grade. Indicating that mt DNA mutation may be an early event during tumorigenesis and lasting for the whole course of tumor progression. mtDNA mutation had a positive relatioship with bcl-2 gene expression , while it had a negative relationship with expression of Bax and caspase.Our study demonstrates that mtDNA could be amplificated and mtDNA mutation be detected successfully in the plasma. It could be used as a valuable tumor marker for tumor diagnosis and a usefulness biological indicator for tumor monitoring.