| ObjectiveColorectal cancer (CRC) is the third most common malignancy in the world. The risk of death is closely correlated to the stage of CRC at the time of primary diagnosis. Sigmoidoscopy significantly improved the detection rate of CRC. However, its wide application is still limited due to high costs and inconvenience. Serum tumor marker which can be easily quantitatively measured, reproducible and cost effective became the hot subjects throughout the world. Plasma/serum markers Carcinoembryonic antigen (CEA) are the only widely used and reliable tumor markers for CRC. That is far from the clinical needs. Thus, specific sera-based tumor markers for early diagnosis and prognosis are needed. Therefore, the purpose of this study is to identify serum biomarkers of CRC. In additon, we further evaluated the potential role of the candidate biomarker ORM2in the differential diagnosis of colorectal cancer and benign colorectal diseases.Method1) We used a quantitative proteomic approach with isobaric labeling (iTRAQ) to examine changes in the plasma proteome of10patients with CRC compared to healthy volunteers. After depleting of12abundant proteins the samples were digested with trypsin and labeled with iTRAQ reagents (114for the CRC group,117for the healthy group). And then MS analysis was performed with MALDI TOF/TOF MS and MICROQ-TOF MS. The differential expressed proteins were identified after DATABASE research. 2) The software MetaCore was used to analyze the relationship of the candidate proteins in the biological network.3) Western blot method was used to confirm the expression of ORM2in CRC patients.41specimens of patient with CRC and41specimens form non-tumorous controls were detected by Western blot to confirm the expression of ORM2from normal controls.4) RT-PCR was performed to validate the differential expression of the candidate between carcinoma and normal tissues on the level of mRNA.5) Enzyme-Linked Immunosorbnent Assay (ELISA) was performed to measure ORM2in serum from419subjects:normal control (n=65), hyperplastic polyp (n=59), inflammatory bowel disease (IBD)(n=62), adenoma (n=53) and CRC (n=180). The CEA was detected using the chemiluminescent microparticle immunoassay technology. Kruskal-Wallis test was applied to determine statistically significant differences, P<0.05was considered as statistically significant. Receiver operating characteristic (ROC) curve was used to evaluate the sensitivity and specificity for ORM2and CEA. All statistical analysis and receiver-operator characteristic (ROC) curve were performed with SPSS software versions13.0for windows (SPSS Inc, Chicago,Ill).Result1) In our quantitative proteomics analysis, we detected76human plasma proteins with more than95%confidence using iTRAQ labeling in conjunction with micro Q-TOF MS. Among this group,13differentially expressed proteins were selected based on1.5-fold over-expression and1.5-fold under-expression in CRC patients, compared to the healthy volunteers.9up-regulated and4down-regulated proteins were observed in the CRC group. The statistical variance of tumor versus normal ratios was within the95th confidence level (P=0.05).2) The network analysis of the differential proteins revealed ORM, complement system and RBP4involvement in CRC.3) When protein expression was measured by densitometer, the median densitometer value of ORM2in CRC cancer tissues was significantly greater than that in corresponding adjacent normal mucous tissues (P<0.01)4) RT-PCR showed that expression of ORM2in CRC was2.65(P<0.01) higher than in normal tissues.5) Median plasma ORM2concentrations were significantly higher in CRC compared to the normal colorectum, hyperplastic polyp, and adenoma (all at P<0.001, respectively). The plasma ORM2level was statistically significantly higher in patients with IBD than in the normal colorectum, colorectal hyperplastic polyp and adenoma (all at P<0.001, respectively) and it was statistically significantly higher in patients with IBD than in CRC (P<0.05). No significant association was found between plasma ORM2concentrations and age, gender, TNM stage or histological grade.Conclusion:Our study for the first time coupled iTRAQ with Q-TOF/MS on plasma specimens of CRC patients to discover potential biomarkers for CRC.13different expressed proteins were identified. Most of them have not been reported as cancer biomarkers that have shown iTRAQ together with Q-TOF/MS is a sensitive, reproducible, robust technique for identifying statistically significant differences of protein expression between the CRC patients and healthy control plasma samples. From the verification of ORM2, it was found that ORM2may be a potential biomarker to distinguish CRC from IBD. In future studies, larger sample populations will be required to confirm these potential biomarkers. In our research, ORM2is firstly identified as a differently regulated protein in CRC patients as compared to normal controls with proteomics screening. However, it requires a lot of bench experiments and clinical tests before ORM2can be used as a reliable biomarker. |
