The Biological Effect Of Regâ…£ On Colorectal Carcinoma

Colorectal carcinoma (CRC) is one of the commonest malignancies which threaten the health of human.In these years, CRC has an increasing incidence and the age of onset becomes younger. People have paid more attention to investigate the pathogenesis of CRC.Adenoma happens in the early stage of CRC, and most of CRC is the result of colorectal adenoma-carcinoma transition. Removal of adenoma can decrease the incidence of colorectal cancer. However,30%～46%patients with adenomas have experienced relapses in more than2.5-7years after the initial treatments. Currently, there is no effective marker to predict the progression or relapse of adenoma. It is vital to investigate the mechanism of adenoma for diagnosis and prognosis of CRC. Many studies focus on the the molecular mechanisms of CRC, but the mechanism of it is still unclear. In1999, our laboratory constructed three cDNA library by suppression subtractive hybridization (SSH), which was constructed from extracts of normal mucosa and adenoma tissue from a single female patient. One of three subtracted cDNA library was A-N library (normal mucosa and adenoma library), and RegⅣ was a significantly up-regulated gene after the analysis of A-N library, which belonged to the regernating gene family.The Reg proteins belong to the C-type lectin superfamily. The member of Reg family is the sectory protein with small molecular weight. They have a similar domain which can bind to sugars with calcium. They take part in inflammation, injury repair, diabetes, malignant tumour and so on. So far, three Reg members have been found in human, including RegⅠ, RegⅢ and RegⅣ. Reg genes are located in the same chromosome2p12except RegⅣ, which is located in chromosome1p13.1-p12. Reg proteins have the similar biological structures and functions.Recently, some studies suggest that RegⅣ is one of the key genes in colorectal tumorigenesis. But there are few researches on its biological roles and effect mechanisms. The purpose of our work is to study its biological roles and molecular mechanisms. Firstly, we chose the proper colorectal cell lines. RegⅣ showed no expression in RKO and HT29, but LoVo and CW2were positive for RegⅣ. RegⅣ expression vector was transfected into RKO and HT29, and RegⅣ-shRNA vector was transfected into LoVo. G418was used for clone selection, and we got the cell clones named as RKO-RegⅣ (RKOR), RKO-Ctrl (RKOC), HT29R-RegⅣ (HT29R), HT29-Ctrl (HT29C), LoVo-RegⅣ-shRNA and LoVo-Ctrl-shRNA, respectively. siRNA of RegⅣ was transfected into CW2, and we named them as CW2-siRNA-RegIV and CW2-siRNA-NC.Subsequently, we studied the biological roles of RegⅣ. Cell proliferation was detected by three methods including cell counting kit-8(CCK-8) assay, EdU assay and xCELLigence system. Cell cycle percentage was measured by flow cytometry. After treatment with5-FU, cell viability was detected by CCK8assay. The cell migration was measured by two methods as Transwell chamber and xCELLigence system. The Transwell chamber with Matrigel was used to detect the ability of cell invasion. After performing gain-of-function and loss-of-function studies, it showed that RegⅣ had no significant effect on cell proliferation compared with the control group. Meanwhile, exogenous recombinant RegⅣ could not stimulate the proliferation of colorectal cancer cells. The results of cell cycle and apoptosis induced by5-FU were similar between the group of overexpression or interference of RegⅣ and the control group. However, migration and invasion abilities of colorectal cancer cells were significantly increased after the inducing by RegⅣ and decreased after RegⅣ knockdown. From the above, RegⅣ could increase the abilities of migration and invasion of CRC cells, but had no effect on the proliferation and apotosis of CRC cells. Then our next work is to study the molecular mechanisms of cell migration and invasion induced by RegⅣ.Comparative proteomics is an important research field in biology. It is available for compare the differential expression and modification of protein between the physiological and pathological conditions. Labeling approaches such as iTRAQ (isobaric tags for relative and absolute quantitation) are a newly developed and quantitative proteomics techniques with high separation efficiency, wide analytical range and low detection limits. It allows multiplexing of up to eight samples to identify the relative and absolute abundance of proteins in different samples within a single liquid chromatography mass spectrometry based proteomics experiment. Therefore, iTRAQ with LC-MS will be a more powerful method in proteome research.We use iTRAQ quantitative proteomics to study the mechanisms of Reg IV in CRC cells. The whole cell lysates of RKOR, RKOC, HT29R and HT29C were labeled with the different reagents of iTRAQ, respectively. We completed the quantative analysis of the identified1985proteins among the cells. Although many differentially expressed proteins were identified, statistic analysis must be perform to verify whether the true differential proteins was or not. The final results were listed as below:11proteins were increased significantly and6proteins were decreased significantly in RKOR compared to RKOC,13proteins were increased significantly and18proteins were decreased significantly in HT29R compared to HT29C. The differential proteins were different between HT29and RKO, because their genetic backgrounds were dissimilar. Considering the metastasis of tumor was the process of multi-genes and multi-pathways, the results from both CRC cells could supply more information and clues for further researches.We analyzied the functions of the differential expression proteins by gene ontology (GO) and the classification of these protein by COG (cluster of orthologous groups of proteins). Moreover, we used the public database KEGG to analyse and identify the important metabolic and signal pathways, which the differential proteins may take part in. Q-PCR was used to detect15and18differently expressed genes in RKORvsRKOC and HT29RvsHT29C, and most of the results accorded with the results of iTRAQ. Then Western blot was uesed to determine the six differently expressed proteins such as MRPL12, MAGED2, DBN1, TIP47, MAPK3and S100A11, and the testing results were consistent with the results of iTRAQ. So the identified differential expression proteins by iTRAQ labled were reliable. All the identified differently expressed proteins may play direct or indirect roles in the process of cell migration and invasion, and they may be the effector molecules of RegⅣ in colorectal cancer cells. It would be useful to elucidate the molecular mechanisms of RegⅣ in colorectal cell lines for further study on these effector molecules and their signal pathways.