| BackgroundColorectal cancer(CRC), including colon cancer and rectal cancer, is one of the most common malignancies in the world, with more than a million new cases estimated to be diagnosed annually. CRC has become the third killer leading cause of cancer. During the past decade, the incidence rate of CRC has rapidly increased in several countries including China, that were previously considered as low-risk region for this disease. In addition, national survey data in China also indicated a significant increase in age-adjusted CRC mortality.It is proved that the carcinogenesis and development of CRC is a multi-stage process which may be initiated by many factors and involved with many genes. Many studies suggested that CRC prognosis is closely related to earlier diagnosis. To date, screening and early diagnosis using approaches such as sigmoidscopy and colonoscopy have been proved to be the most effective means to reduce CRC incidence and mortality.However, although these approaches have relatively high sensitivity and specificity, they are highly invasive and expensive, making it difficult for them to be applied to the general population.With the development of molecular biology, genetics and biotechnology, our country has achieved considerable progress in terms of basic and clinical research and prevention of CRC recently. So far, it remains unclear for the pathogenetic cause, recurrence and metastasis of CRC. To screen the high-risk populations (over 40 years old or have CRC family history or have intestinal polys) can early detect CRC, but it has no obvious clinical symptoms at the onset of CRC. The specificity of carcinoembryonic antigen (CEA) still remains undesirable level, and sigmoidscopy and colonoscopy are not to be applied to the general population. At present, a non-invasive and cost-effective approach is to be badly in need of early screen of CRC. Therefore, it is necessary to develop novel biomarkers, in order that are most likely to develop CRC for more intensive and targeted screening and preventive interventions. Mitochondria play important roles in cellular energy metabolism, free radical generation and apoptosis. Recently, mitochondrial DNA(mtDNA) has been sequenced completely. These make great sense for discussing the correlation between mtDNA and cancer.The number of human mitochondria in a cell ranges from several hundred to more than 10000 copies, and each mtDNA contains 2 to 10 copy numbers. Human mtDNA is a maternally inherited genome consisting of a 16569 base-pair (bp) circular double-stranded DNA molecule. There are no introns in mtDNA.Unlike nuclear DNA (nDNA), mtDNA is less protected by histones and appears to have less-efficient repair mechanisms, which makes mtDNA particularly susceptible to reactive oxygen species (ROS) and other genotoxic damage and become a cellular target for carcinogenesis. For these reasons, mtDNA have several outstanding features including higher content, mutation, polymorphism of mtDNA and its maternal inheritance. The mtDNA mutation (including alteration of mtDNA content) is closely correlated with cancers occurring and development in a tumor-specific manner. The clinical studies used of somatic tissue samples have shown the significant mutation and alteration of mtDNA, and the abnormal variations also exist in leukocyte of cancer patients. However, it remains unclear how mtDNA play a role in tumor carcinogenesis and progression.Thus, our research aim to find the characteristic of alteration of mtDNA content in CRC patients'peripheral blood lymphocytes (PBLs) and the correlation between CRC patients'PBLs and normal colorectal tissues. We further investigate whether the easily procured blood specimens could be used as surrogates for tissues to research the correlation between alteration of mtDNA content and CRC risk and prognosis. We also want to identify whether mtDNA content in PBLs could be used as a risk and/or prognosis predictor for CRC. It can be helped to early detect CRC and to prolong the CRC patients'life expectancy.Method1. Used a case-control study, a total of 320 incident cases aged 22-87 years old with histologically confirmed primary CRC were consecutively recruited from Department of Gastroenterological Surgery in the Tangdu Hospital that is affiliated to the Fourth Military Medical University, Xi'an, Shaanxi,and matched 320 healthy people aged 22-88 years old as control. All cases were first diagnosed as CRC, and had no prior chemotherapy or radiotherapy. There were no age, sex, or disease stage restrictions for case recruitment. The 320 healthy controls without previous cancer history (except non-melanoma skin cancer) were recruited from individuals who visited the Tangdu hospital for physical examination, during the same time period as the case enrollment. The controls were frequency-matched to the cases on age (±3 year), sex and residential areas. All participants were Han Chinese. 2. Peripheral blood of 320 CRC patients and 320 healthy controls were collected, 10 adjacent normal colorectal tissues with the histological confirmation were randomly selected. Genomic DNA was extracted from tissue and peripheral blood by using E.Z.N.A. tissue DNA Kit (Omega Bio-Tek ), then stored in refrigerator with -40℃.3. The mtDNA content was determined in PBLs-derived genomic DNAs of all participants, and mtDNA content in normal colorectal tissues and their paired PBLs of 10 random selected CRC patients were also determined.4. After signed informed consent was obtained from each individual, all participants were collected demographic and personal data, including age, gender, smoking history, alcohol consumption, education status, body mass index (BMI), dietary patterns, family history of cancer, environmental exposures, physical activity, use of non-steroid anti-inflammatory drugs (NSAIDs), and hormone replacement therapy(HRT). The clinical data of all CRC patients were collected, including operation date, tumor position, tumor histology, tumor differentiation, clinical stage, and chemotherapy. After surgery, a standard follow-up was performed to collect the postoperative information of CRC patients, including postoperative treatment, recurrence, metastasis, and death.5. SPSS 16.0 statistical package (SPSS, Chicago, IL) were used for the statistical analyses of the correlation of mtDNA content of CRC patients between PBLs and normal colorectal tissues, and the relationship between mtDNA content in PBLs and CRC risk and prognosis. The association between risk and prognosis of CRC and mtDNA content was estimated using odds ratios (ORs), hazard radio (HR) and 95% confidential intervals (CIs). All P values were based on 2-sided tests. A probability level of 0.05 or 1.0 was exclusive of CI was used as the criterion for statistical significance. Result1. Distribution of general characteristics in all participants. The CRC cases and healthy controls were well-matched on sex (P = 1.00) and age (58.42±13.12 vs. 58.16±13.53 years old, P = 0.807). There were no statistically significant differences between cases and controls in terms of alcohol consumption (P = 0.395), education level (P = 0.169), BMI (P = 0.632) and NSAIDs use (P = 0.477). Cases had more ever smokers (39.37% vs. 32.81%, P = 0.084) and higher pack-years than controls (26.2±14.94 vs 20.29±13.05 pack-years, P = 0.002). The mean coefficient variation (CV) was 6.9% (range, 3.9 % to 9.1 %) and 4.2% (range, 2.4 % to 6.9 %) for inter-assay and intra-assay CV, respectively.2. Distribution of mtDNA content. The mtDNA content in PBLs was significantly higher in CRC cases (median: 1.03; range: 0.46- 3.37) than controls (median: 0.86; range: 0.48-3.06) (P < 0.001). We further analysed the mtDNA content stratified by host characteristics found that no modulating effect of sex, age, BMI, smoking status and alcohol consumption on mtDNA content was identified in both cases and controls (P value range, 0.151 to 0.909).3. The correlation analysis of mtDNA content between PBLs and normal colorectal tissues. Pearson's correlation analysis was used to estimate the relevance of mtDNA content between 10 CRC patients'PBLs and paired normal colorectal tissues. The results showed that there was a significant positive correlation for mtDNA contents between normal colorectal tissues and paired PBLs (r = 0.659, p = 0.038).4. The correlation analysis between mtDNA content and CRC risk. We performed unconditional logistic regression analysis to assess the association between mtDNA content and CRC risk. We found that individuals with high mtDNA content had a significantly increased risk of CRC (adjusted OR = 2.04, 95% CI = 1.48 to 2.82) compared to those with low mtDNA content. We further categorized the subjects into four groups based on the quartile values of mtDNA content in the controls. We observed a significant dose-response relationship between CRC risk and mtDNA content in PBLs (P<0.001). The association between increased CRC risk and high mtDNA content was not modulated by major host characteristics as indicated by the stratified analysis.5. The relevance analysis between mtDNA content and CRC prognosis. 320 CRC patients had follow-up information, we excluded 43 patients, including 6 patients who did not undergo surgery or only received palliative operation, 37 patients who failed follow-up. Finally, 277 patients with resected colorectal adenocarcinoma were included in the prognosis study. The Cox multi-variable regression analysis showed that there were no obvious relationship between CRC prognosis and age, sex, smoking, alcohol consumption, tumor position, and education level by using Cox regression model method. But clinical stage and tumor differentiation were significant related to CRC prognosis. The higher stage had a worse overall survival rate (use stage 0/Ⅰas reference, from stageⅡtoⅣ, adjusted HR: 6.81, 11.98, 25.48), and the poor differentiation produced a worse prognosis (use well differentiation as reference, moderate and poor differentiation adjusted HR: 1.55, 2.53). Postoperative adjuvant chemotherapy had a significantly decreased risk of death (adjusted HR = 0.57) compared with patients without chemotherapy. mtDNA in PBLs was significantly related to CRC recurrence (P = 0.008) and death (P = 0.007). Patients with higher mtDNA content in PBLs conducted a worse prognosis (adjusted HR of recurrence and death: 1.87, 2.01) compared with those who had lower mtDNA content. Kaplan-Meier analyses showed that, compare to patients with lower mtDNA content, patients with higher mtDNA content had a poor survival rate (Log rank P = 0.002).Conclusion1. There was a significant positive correlation for mtDNA contents between normal colorectal tissues and paired PBLs of CRC patients. These indicated that mtDNA content in PBLs can be used as a surrogate of mtDNA content in colorectal tissue to detect its variation.2. Increased mtDNA content in PBLs of CRC patients was significantly related to CRC risk, and CRC risk can be evelated by higher mtDNA content. There was a statistically significant dose-response relationship between higher mtDNA content and increased CRC risk.3. mtDNA content in PBLs of CRC patients was significantly relevance to CRC prognosis. Patients with higher mtDNA content in their PBLs had a poor survival rate. From these results, we suppose that mtDNA content in PBLs might be used as a risk and/or prognosis predictor for CRC. |
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