Mitochondrial Genome Instability In Sporadic Colorectal Cancer

Background:Colorectal cancer remains the third cause of overall malignant incidence and mortality in worldwide.According to the data provided by the researchs on Cancer,The Asian region,especially in developed areas,The incidence of colorectal cancer is also growing rapidly close to the West Country. With China's rising standard of living,changed eating habits,the overall incidence rates are also on the rise.It is a serious threat to people's health.So looking for the pathogenesis of colorectal cancer becomes a hot spot.The process of tumor growth requires a large amount of energy to supply unlimited cell proliferation,Cellular energy,which is mainly from mitochondrial aerobic oxidation and glycolysis.Previously researchers considered that because of tumor rapid growth,long-term hypoxia status,glycolysis in the tumor can play a leading role.In recent years,researchers think these two ways of energy supply in tumor play equal roles.Mitochondria are the only genes outside the nuclear in eukaryotic cell.Somatic mutation rate of it was presumed to be 10 to l00 times higher than that of nuclear.In recent years,with the deep understanding of mitochondrial inducing cell carcinogenesis,the role of mitochondrial DNA mutations has become one of the hot spots in cancer research.MtDNA mutations were reported in different types of carcinoma and carcinoma cell lines.Reported sequence changes include point mutations,multiple deletions and microsatellite instability in coding and noncoding regions.However,little is know about mitochondrial DNA large deletions,microsatellite instability and alteration of the copy number in human colorectal cancer.The further study of mitochondria will provide a new basis on the occurrence of colorectal cancer.Objective:(1)To study the 4977bp deletion and D-loop mutation of mitochondrial DNA in colorectal cancer,adjacent normal and control normal intestinal tissue and coding region mtMSI frequency and the relationship between it and clinical indicators such as age,histological pathology type and nuclear MSI.(2) To develop a precise assay based on PCR to determine the copy number of mtDNA in colorectal cancer and elucidate if there is a link between alteration of the copy number and prognosis of patients.Methods:(1)50 matched colorectal cancers/adjacent normal and 20 normal intestinal tissue samples were analyzed by long PCR technique.(2)Mitochondrial DNA D-loop of 50 matched colorectal cancers/adjacent normal and 20 normal intestinal tissue samples were analyzed by long PCR and sequencing technology.The mtDNA of the D-loop was divided into three sections to sequencing,the results comparison to Cambridge sequence were edited by BIOEDIT to analyze mutation frequency and distribution.According to the data from the Mitomap web,sequence changes were judged as polymorphism and mutation.(3) Using STR method to scan the five microsatellite loci of the mitochondrial coding region and the two microsatellite loci of nuclear DNA.Analyzing the relationship between mtMSI and age,pathology-histological types,nMSI.(4) 60 cases paraffin specimens of colorectal cancer and the corresponding normal margin in 2005 stored in our hospital's pathology department were cut at 10μm thickness 5 slices.DNA was extracted from microdissected sections of these slices. The nuclear geneβ-actin was chosen as the internal reference for quantifying mtDNA copy number and a marker of diploid genome content.ND1 represents mtDNA copy number.A precise assay based on fluorogenic real-time quantitative PCR was developed to compare the relative abundance of mtDNA with nuclear DNA.Chi-square test and Non-parametric test were used to analyze the relationship between copy number and age,gender,pathological type,clinical stage,metastatic lymph nodes.Kaplan-Meier and log-rank methods were used to estimate the survival function.Results:(1)Mitochondrial DNA 4977 bp deletion was detected in 10%(5/50) of colorectal cancer,18%(9/50)of adjacent normal and 15%(3/20)of normal intestinal tissue samples.The frequency of mtDNA⁴⁹⁷⁷ deletions increased with age in colorectal cancer,adjacent histologically normal and normal intestinal tissue samples.Tissue samples were divided into high and low age groups,and the incidence of the mtDNA⁴⁹⁷⁷ deletion in high age group was significantly higher than in those of low age group(P＜0.01).(2) Among the mtDNA D-loop region of 50 colorectal cancers,16cases of carcinoma patients were found 13 mutations.Mutation types are:9 point mutations, 3 microsatellite instabilitys,one deletion mutation.The mutation number of D303 is 11,3 are homogenicity,8 are heterogeneity;Two happened in D514,Two happened in D16184,both are heterogeneity.Three of these mutations occurred in the heavy chain of the replication origin and another four point mutations occurred in the hypervariable region I.Mitochondrial DNA D-loop region mutation rate of colorectal cancer is 32%(16/50)and 20 normal control intestinal tissue found 2 mutations(16184,16280 locus)(10%).The difference of mutation rate between cancer and normal tissue has statistical significance(P＜0.001).Nucleotide polymorphisms were found in 52 sites.(3) 15 cases of mtMSI at coding region were detected,the occurrence rate is 30% (15/50) and nMSI were detected in 11 cases,the incidence rate was 22%(11/50).A fight colon cancer patient is positive for all loci,nMSI was detected in 5(10%) at locus BAT25,9(18%) at locus BAT26;mtMSI was detected in 6(12%) specimens at locus ND1,in 2(4%) at locus ND2,1(2%) at locus COâ…¢,and 2(4%) at locus ND5-1,8(16%) at locus ND5-2.(4)The incidence of mtMSI at control and coding region was 38%(19/50) had no relationship with gender,age and tumor location and histological type(P＞0.05), but there were significant differences between mtMSI and nMSI(P＜0.05).(5) The mean copy number of mtDNA(108.60±20.11)in the tumorous tissues was significantly lower than that(153.68±25.72)of the corresponding non-tumorous tissues of these patients(p＜0.001).For statistical analysis of the mtDNA copy number value,the patients were divided into two groups using a cutoff level ot 0.72,which was the median value of the T/N ratio.No significant correlation was found between mtDNA copy number and other variables including age,gender, pathological type and clinical stage,however,twelve of 30 patients with a low mtDNA copy number value had metastatic lymph nodes,while only five of 30 cases with a high mtDNA copy number value were detected.Patients had a tendency to have a shorter tumor-free survival time in low mtDNA copy number value than in high group when assessed by Kaplan-Meier curves,but it is not significant(P=0.089).Conclusions:(1)Those results did not support the notion that the mitochondrial DNA 4977-bp deletion plays a major role in colorectal cancer.The accumulation of mtDNA⁴⁹⁷⁷ deletion associated with aging,which reflect the roles of the environment and the age factor.(2)This study detected 52 nucleotide loci polymorphism variation and 32%(16/50) mutation rate.Those show that D-loop region is a highly polymorphic fragment,at the same time reflects a high incidence of somatic mutation.The genetic instability of mtDNA non-coding region may lead to abnormal replication,transcription those end in tumor formation.(3)Base insertion or deletion is a common cause of the mitochondrial microsatellite instability in CRC.The mutation of mismatch repair gene affects not only the nuclear gene but also the mitochondrial genome.So mtMSI may play a role in the occurrence and development of colorectal cancer.(4)The copy number of mtDNA decrease significantly in the tumour tissues of the colorectal cancer patients as compared with that in the corresponding non-tumorous tissue.The copy number was negatively correlated with the metastatic lymph nodes,which may imply that the decrease of copy number is also related to tumor invasiveness.The patients with high copy number have longer tumor-free survival time than patients with low copy number.Therefore,this reduction of mtDNA copy number may well serve to identify CRC patients with a poor prognosis through further study.