| Colorectal cancer(CRC) is the most common gastrointestinal cancer the fourth leading cause of cancer-related death in the world. The incidence of colorectal cancer in Chinese people increased year by year due to smoking, drinking, the change of diet, and showed younger trend. In the treatment of colorectal cancer, there are cytotoxic agents and targeted agents which efficacy has been proven. And the targeted agents, mainly include anti-VEGF and anti-EGFR antibodies, have become the most promising treatment for the patients with advanced colorectal cancer. But only some patient benefited from the targeted agents because of their limitations. To discover the new therapeutic target for individual treatment remains one goal of many researchers.In the process of occurrence and development of tumor, the cells can produce a series of changes in protein expression. Proteomic analysis technology can be used to detect the protein expression spectrum of the tumor cells, which contribute to understand better the mechanism of tumorigenesis, and speed up the process of finding the tumor markers and therapeutic targets. The emergence of the protein chip provides a powerful technology platform for cancer proteomics research. As a kind of protein chip,antibody microarrays(abm) has the characteristics of miniaturization, integration, high flux, and can be used to detect a particular physiological or pathological process related proteins expression abundance. At present, abm is mainly used in signal transduction, proteomics, cancer and other diseases related research.Although Japanese scholars have been applied abm(AAH-BLM-1-2, raybio IT company)to detect the proteins expression of colon cancer and rectal cancer respectively, but they did not verify the results and not select some representative candidate biomarkers to further explore their Clinical Significance by immunohistochemical analysis.The purpose of this study is to detect the differentially expressed proteins beteween tissue samples from primary colorectal cancer and normal colorectal mucasa by using antibody microarray (including 274 cell function protein),which could identify novel protein markers for colorectal cancer early diagnosis, evaluation of therapeutic effect and prognosis and for the treatment to provide a new target protein. Among the upregulated and downregulated proteins with statistical significance in CRC tissues, one proein-CCL5 selected as the representative candidate biomarker, was validated by enzyme-linkedimmunosorbentassay (ELISA). The Clinical significance of CCL5 expression for tumor progression was investigated in an independent primary cohortn by using immunochemitry. This study is the first to demonstrate a significant correlation between the expression of CCL5 and the and adverse pathological characteristics (tumor stage, node metastasis) in CRC.Objective1. The purpose of the present study was to identify novel candidate biomarkers in primary colorectal cancer using an antibody microarray (abm).2. Among the upregulated and downregulated proteins with statistical significance in CRC tissues, the exprssion of one protein selected as the representative candidate biomarker was validated by enzyme-linked immunosorbentassay (ELISA).3. To investigate the expression site of the selecting protein in colorectal cancer, and the correlation of this protein with clinicopathological features by immunochemitry.Materials and methods1. Four pairs of tissue samples from primary colorectal adenocarcinoma and normal colorectal mucosa were analyzed by Reybiotech AAH-CYT-G6-G10 analysis of microarrays and differentially expressed proteins were identified.Differentially expressed proteins between normal colorectal mucosa and CRC were selected based on the criteria that the difference of light signal intensities beween two samples was statistically significant (P＜0.05). Signal intensities for each spot were calculated by subtracting the signal intensity of negtive control spot.2. Among the upregulated and downregulated proteins with statistical significance in CRC. RANTES/CCL5 was selected as the representative candidate biomarker. Furtherly, the expresson of CC5 was validated ELISA in 38 pairs tissue samples from primary colorectal cancer and normal colorectal mucosa.3. The expression of CCL5 was detected in 60 cases of patients with CRC.To validate the prognostic values of the selected candidate protein-CCL5, immunohistochemical studies were performed on tissue specimens from 60 patients who had been diagnosed with primary colorectal cancer. A HRP-labeled polymer detection system was used with an Fast-Red chromogen. Scores were independently assessed by four pathologists.Rusult1.25 differently expressed proteins were found between tissues of colorectal cancer and matched normal colorectal mucosa, including 14 up-regulated and 11 down -regulated proteins.2. The ELISA result showed that the level of CCL5 expression in tissue of colorectal cancer Were higher than that in normal colorectal mucosa..3. CCL5 was highly expressed on the cytoplasmic membrane of CRC cells, whereas its expression was weak or absent in normal colorectal epithelium. Positive CCL5 expression was significantly associated with adverse pathological characteristics, including lymph node metastasis and higher T stage, whereas there were no statistically significant relations between high expression of CCL5 and other clinicopathological factors.Conclusion1.This study demonstrated the potential applicability of abm profiling for identifying novel biomarkers in CRC.2. The association of high CCL5 immunostaining with nodal metastasis and tumor stage supports involvement of CCL5 in tumour progression.3. CCL5 is a promising target that may help in understanding the pathogenesis of CRC.4.CCL5 as a potential molecular marker of metastases and as a new targeted therapeutic target warrants Further molecular in vitro and in vivo studies. |
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