| Background and ObjectiveUlcerative colitis (UC), as one category of inflammatory bowel disease, is a chronic, spontaneously relapsing, debilitating idiopathic, non-specific inflammation. UC is characterized by crypt abscess and ulceration of the colon mucosa and submucosa, the key features are diffuse mucosal inflammation that extends proximally from the rectum to a varying degree. The main clinical manifestations of UC are diarrhea, abdominal pain and bloody mucous stools with pus. UC is an idiopathic chronic disease of the colon characterized by periods of active disease followed by periods of remission. UC can be found in all range ages, also chidren and elder, especially20-40years old. There is no significance of incidence between male and female. The pathology feature are the diffuse inflammation of mucous membrane and the diffuse infiltration of lymphocytes, plasma cells and mononuclear cells within lamina propria, the activity has a large number of neutrophils and eosinophils infiltration, inflammation can occur fossae and crypt abscesses. In the1960s the British scientist Morson sorted it with crohn’s disease depends on pathological features and defined it as an independent disease. UC is very common in European and North American countries, the investigation from20centres in European indicated that the incidence of UC is11.2/105population/year, recently there is convincing evidence of rising trend of UC in oriental countries which have lower incidence in old days.1981-2000, a total of10218reported cases of UC in our country, which increased3.1times in a decade. Temporary lack of large-scale epidemiological investigation in our country, but a rough estimate incidence of UC as of11.6/105, and there is evidence that a rising trend year by year. Moreover, ulcerative colitis is a kind of precancerous lesions, associated with colon cancer, and UC usually has a long duration, with different pathological changes degree and unknown etiologylt recrudesces time after time and relates to colon cancer. Because UC recurs frequently and is hard to cure completely, it ranks among refractory diseases of modern times by world health organization (WHO). To achieve the purpose of cure UC, the early detection and the pathogenesis research are very important.Although the research methods which deep into the molecular biology, gene level and protein level, makes the research of the pathogenesis of ulcerative colitis has developed by leaps and bounds, we must be clear of ulcerative colitis is multiple factors involved in complex diseases, single gene or protein markers cannot fully and accurately reflect its complex pathogenesis. So we need to use the genome, transcriptome and proteome high throughput technologies to search the compound marker genes or proteins, in order to more accurately and sensitively predict disease, and explain the molecular mechanism of development. We all know that the genome level research, mainly concentrated in the micro satellite instability and gene amplification, missing. Transcriptome research is focused on detection of Gene expression changes in mRNA level, high flux to test samples of tissues or cells in Gene mRNA level, and to establish the Gene expression patterns, so as to determine the amount of disease related genes expressed in a set of (Gene signature), used for disease evaluation and prognosis. Compared with the genome level of research, Gene signature is more practical. While the proteomic not only investigate the expression of protein products of genes, but also pay attention to the detection of protein modification after translation and its influence on protein function. The three levels of study are not isolated, which reflect the abundance of differential gene and the expression change, three levels of the research results can confirm each other, complement each other.Early we first used two-dimensional electrophoresis and mass spectrometry, compared12cases of ulcerative colitis and12cases of normal tissue protein expression spectrum, and identified30differential expressed proteins. Using bioinformatics methods, we further found11kinds differential proteins related with P38MAPK pathway, and we combined the11kind proteins with the key proteins of this pathway as a differential protein cluster. We used Western Blot technique to validate the correlation of differential protein cluster with ulcerative colitis in the above sample. And then chose two kinds of protein from the protein clusters, Galectin-3and MAWBP, we used immunohistochemical technique to test its abundance in58cases of ulcerative colitis and normal samples. We found that the expression of Galectin3and MAWBP significantly reduced in ulcerative colitis group (χ2=31.404, P=0.000, χ2=20.541, P=0.000) and specifically associated with the degree of inflammation (x2=12.315, P=0.002, χ2=8.126, P=0.018).In this study, we further explored the correlation and the regulatory mechanism of P38MAPK signaling pathway with ulcerative colitis on the basis of the preliminary test results, and further expand the sample size and investigated the the possibility of protein signature, extracted from differential protein cluster, as the biomarker of unicerative colitis. We were very interested in the MAWBP, a new protein of the protein signature, which was closely associated with ulcerative colitis.So we used genomics combined with a variety of molecular biology techniques to investigate its biological function and its relationship with ulcerative colitis. We also further analyzed the complicated mechanism of ulcerative colitis delay, using genomics and found an important tumor related genes, and thus provides the candidate biomarkers of precancerous lesions in ulcerative colitis and promising new treatment targets.Methods1. Using Western Blot and ELISA technology, we verify the previous proteomics differential protein clushter regulated by P38MAPK signaling pathway in the macrophage cell line RAW264.7, then we used immunohistochemistry technique to investigate the expression of three different proteins cluster, phosphorylated P38, Galectin3and MAWBP in118cases samples and analyzed the correlation with clinical features of ulcerative colitis and the possibility of compound biomarkers.2. Using Microarray and molecular biology method to reveal the role, function and molecular mechanism of the differential protein MAWBP in ulcerative colitis. First we detect the expression and localization of MAWBP binding protein (MAWD) by immunohistochemistry and Western Blot in ulcerative colitis, and then we confirmed whether they can combine with each other by immune co-precipitation experiment. Then we construct the eukaryotic expression vector of MAWBP and MAWD, and respectively or simultaneously transfect into Caco-2cells, we analyze the gene expression profile through Microarray technology, and validate the differential genes by Real-time PCR and Western Blot. Finally we knock down MAWBP and MAWD expression by RNA interference technology and further from the opposite direction to verify the correlation of MAWBP and MAWD with ERK pathway.3. Using DASL technology to analyze the gene expression profile of active ulcerative colitis and inactive ulcerative colitis at transcription level, choose the same patient at the same site active and inative stage endoscopic specimens, a total of29cases, bioinformatics analysis of differential genes, further validated by Real-time PCR and IHC in differential gene expression.4. Statistical analysis:we use SPSS13.0software to do all the data analysis. Pearson chi square test and Fisher’s exact probability method to analyze the relationship between the pathological characteristics of protein expression and disease. Analysis of variance and subsequent multiple comparison method based on the analysis of variance to analyze Real-time PCR results and differential genes and proteins of composite omics, multiple test P values need to correct with Bonferroni method. P<0.05was considered statistically significant; We used Pearson correlation test to detect the correlation of two results, r>0.5was considered strong correlation.Results1. A differential protein cluster involved in p38mitogen-activated protein kinase (MAPK) pathway was deduced and validated by Western blot. Furthermore, three proteins elicited from the protein cluster, phosphorylated p38, MAWBP and galectin-3, as a molecular signature, was analyzed by immunohistochemistry. Increased expression of P-p38and down-regulated MAWBP and/or galectin-3were detected in UC compared to normal samples (χ2=41.509, P<0.001; χ2=62.668, P<0.001;χ2=6%.991, P<0.001), and classified UC risk with high sensitivity (94.83%) and specificity (98.33%). These results indicate that molecular signature of P38MAPK pathway might be a potential biomarker for evaluating UC risk.2. Bioinformatics analysis indicated that MAWBP is MAWD binding protein; immunohistochemistry and Western Blot showed MAWD also decreased expressed in ulcerative colitis tissues such as MAWBP. Immune co-precipitation experiment showed that the two proteins can be combined each other to form compounds. Real-time PCR and Western Blot showed MAWBP and MAWD eukaryotic expression vector successfully transfected into Caco-2cells. Bioinformatics analysis revealed MAWBP and MAWD can result in ERK1pathway activation, oncogene TPX2high expressed, and inflammation factors and chemokines can be suppressed. Real-time PCR verified the inflammation factor IL8, TPX2TSC1, CXCL2, IFIT1and oncogene TPX2(P<0.05). Microarray and Real-time PCR results strong correlated (r>0.72). Western Blot validated that MAWBP and MAWD can activate ERK pathway, they can inhibit ERK pathway through knock down MAWBP and MAWD expression by RNA interference RNA3. We applied DASL to elucidate the differential gene profile of UCative and UCJnactive. A total of909up-regulated differential genes were obtained and823down-regulated differential genes. We categorized up-regulated genes into several groups, including inflammation, colorectal adenoma related oncogene, EMT, and the down-regulted genes involved in the PPAR pathway, tumor-suppressor genes related to colorectal adenoma. We validated colon cancer related oncogenes MMP1and MMP3and CHI3L1and CHRDL2, tumor suppressor genes ABCG2and PLA2G12B by Real-time PCR in UC_active and UC_inactive which have significant difference (P<0.05),we further found that MMP1and CHI3L1and ABCG2had sigficant difference between UC and normal and colon caner (χ2=8.418, P=0.015;χ=6.995, P=0.030; χ2=6.285, P=0.043), which indicatead that UC had high risk of canceration. Combination of proteomics and Western Blot, Real-time PCR results found that mesenchymal markers Vimentin up-regulated in UC, epithelial markers E-cadherin down-regulated in UC.Conclusion1. The molecular signature obtained from the integrate proteomics consisting of three differential proteins, the phosphorylated P38, Galectin3and MAWBP, might be a potential biomarker for evaluating UC risk.2. Using the integrate genomics found that MAWBP and its binding protein MAWD can activate the ERK pathway and oncogene TPX2, and inhibit the role of inflammation in ulcerative colitis.3. Using the integrate genomics discovered oncogenes and tumor suppressor genes in ulcerative colitis without carcinogenesis have differential expression.4. Applying integrate proteomics and genomics found that ulcerative colitis might have the EMT process. |
