Dynamic Transcriptome And The Molecular Mechanisms Of Nonresolving Colitis-Associated Carcinogenesis

[Background]Nonresolving inflammation is associated with tumor initiation, promotion and progression. Colorectal cancer (CRC)—the third most common cancer in the world—has been endangering human health seriously. Nonresolving colitis, such as ulcerative colitis (UC), is at increased risk of developing CRC, and is associated with the site, extent and duration of inflammation. Evidence is mounting to support a similarity between colitis-associated cancer (CAC) and CRC, which is a multi-gene, multi-step and multi-stage process. However, different from "adenoma-carcinoma" sequence in CRC, colitis-associated carcinogen-nesis experiences a unique sequence of "inflammation-dysplasia-carcinoma". Untill now, the research about the mechanism of CAC has just begun. The dynamic omics study is still lacking, and the recognition of its molecular mechanism and the regulation network is poorly understood. In the present study, we aimed to find the molecular events and its regulation network of the "inflammation-cancer chain" in both transcription level and protein expression level. We performed gene expression array experiments combined with bioinformatic analysis, serum inflammatory antibody array experiments, real-time quantitative PCR and immunohistochemistry detection, based on a chemical induced nonresolving colitis and colitis-associated colorectal cancer mouse model we had established. We dynamically analysed and summarized the changing regularity of molecular events in the process of "inflammation-dysplasia-cancer" sequence, to provide the theoretical basis for the patho-genesis, clinical diagnosis and treatment of nonresolving colitis and CAC. [Establishment of a mouse model of nonresolving colitis and colitis-associated colorectal cancer]We established a nonresolving colitis-CAC mouse model by administration of chemical reagents, azoxymethane (AOM) and dextran sulfate sodium salt (DSS). This model mimicked human UC and CAC closely, for it has the same characteristics as human's:(1) the same clinical manifestations:diarrhea, stool occult blood, mucopurulent bloody stool, and weight loss;(2) the similar pathology:varying degrees of inflammation, erosions and ulcers were observed with a large number of inflammatory cells infiltrated into the large bowels after DSS administration; later, as the3cycles of DSS administration, nonresolving inflammation established, and the "inflammation-dysplasia-adenocar-cinoma" sequence presented in the gut. What's more, the dysplasia had a plat form, and multifocal of dysplasia and cancer were presented in the region with more severe inflammation;(3) the same gene expression profiles as human beings. The AOM/DSS induced UC and CAC mouse model we have established has scientific application value. It can provide guarantee for the successor examinations.[Gene expression profiles in different pathology stages of nonresolving colitis-associated colorectal cancer]The transcription level embodies the genome genetic information. In order to fully reveal the gene expression profiles at each stage of CAC, we performed gene expression array experiments on the "normal-inflammatory-dysplastic-cancerous" intestinal mucosa. We found that gene expression at every stage varied with a certain rule. In the early period of the inflammatory phase, molecular change turned to be the most significant. There were2245and2294gene probesets upregulated and downregulated in inflammatory mucosa compared with normal mucosa, respectively, formed a "transcriptional storm". A variety of clustering analysis showed that the inflammatory samples strictly clustered, and hold an absolute dominant position by the principal component analysis. Gene ontology (GO) enrichment network analysis found the predominate differentially expressed gene sets in this phase were the gene sets participate in inflammation, including cytokines, chemokines, adhesion molecules, angiogenic factors and the matrix reconstruction factors, apoptosis inducing factors, survival and proliferation factors, and so on. These gene sets expression changes eased up in the dysplastic and cancerous phases, which indicated that the body was in an active defense state at the beginning, but gradually tolerance to inflammation and tumor cells. These results also imply that the tumor microenvironment has a role in the transformation of the immune cells. The early upheaval may pave the way for the later disease progression, and determine cells fate. In the cancerous phase, part of inflammatory factors were still active, but the gene sets such as cell proliferation promotion, apoptosis inhibition, angiogenesis, and matrix degradation occupied an leading position, which fully reflected the malignant behaviors of colorectal cancer cells.[Dynamic activation of the key pathways:the link between inflammation and cancer]We first time report that the NF-κB, STAT3, p38MAPK and Wnt/β-catenin signaling pathway have a periodic activation in the process of colitis-associated carcinogenesis, through gene expression analysis and immunohistochemistry detection. The canonical NF-κB pathway (p65) and STAT3signaling pathway were persistent activated during the "inflammation-dysplasia-carcinoma" sequence. p65, p-STAT3and their target gene coding proteins Bcl-xL and COX-2were upregulated. The alternative NF-κB pathway (p52) was activated only in the inflammatory cells, especially in the inflammatory phase, while downregulated in the dysplastic and cancerous phases. p38MAPK signaling was activated in the inflammatory phase and dysplastic phase, while inactivated in the cancerous phase. Wnt/(3-catenin signaling pathway was activated from the cancerous phase. These findings indicated the canonical and alternative NF-κB pathway, STAT3and p38MAPK pathway may have an important role in the initiation and maintenance of the inflammation. They involved in the inflammatory cells chemotaxis and proinflammatory factors releasing. The persistent inflammation induced by the canonical NF-κB pathway, STAT3and p38MAPK pathway could play a role in the transformation of the intestinal epithelial cells. Activation of the above signaling in the cells of the microenvironment brought about the persistent cytokine production and promoted the transformed cells survival and growth. At last, with the Wnt/β-catenin signaling pathway activation and p38MAPK (regarded as a tumor suppressor) inactivation, the balance between pro-tumorigenesis and anti-tumorigenesis was broken, and tumor established and stable development in the background of the inflammation induce by the canonical NF-κB and STAT3pathway.[The key nodes of dynamic interaction networks during nonresolving colitis-associated carcinogenesis]Combined with gene expression array data analysis and serum inflammatory antibody array experiments, we found the key nodes of dynamic interaction networks during nonresolving colitis-associated carcinogenesis. In the inflammatory phase, IL-1(3, IL-6, COX-2and CEBPB were in the center of upregulated molecular network, while CYP family members, occludin and HNF4a were in the center of downregulated molecular network. In the dysplastic phase, CD44and VCAM-1were the key nodes of the upregulated molecular network, while CYP and GSTM were the key nodes of the downregulated molecular network. In the cancerous phase, IL-23a, CXCL2, IL-1β, LCN2, TIMP1, MMPs and TCF2were the key nodes of the upregulated molecular network, while CYP and ALDH family member were the key nodes of the downregulated molecular network. These key molecules correlates with many other differentiation expressed molecules, and related to NF-κB, STAT3, p38MAPK and Wnt/β-catenin signaling pathway closely, forming the dynamic regulatory network, which promote the nonresolving inflammation induced malignant trans-formation.In summary, establishment of the gene expression profiles during the "nonresolving colitis-dysplasia-colorectal cancer" sequence by using a mouse model, and validated primarily in the protein level, provide us a valuable theory and experiment evidence to further explore the underlying molecular mechanisms and its regulatory networks. In the present study, we found the dynamic regularity of the genome-wide transcription. The "transcription storm" in the early phase of inflammation seems to determine the end of disease. Through bioinfor-matic analysis and immunohistochemical validation, we first time report the NF-κB, STAT3, p38MAPK and Wnt/β-catenin signaling pathway have a periodic activation, which was confirmed by the serum inflammation antibody array detection. This finding indicates the periodic activation of key signaling pathways have a promotion role in the inflammation-cancer process. Although there is a high homology between mice and human, to expand the results of this research have to be confirmed by clinical experiments. Data integration from both human and the animal model samples will provide new avenues to the early diagnosis and treatment of UC and CAC. What's more, the conclusions of the study remind medical workers should pay high attention to the prevention and treatment of chronic inflammation, to prevent it from malignant transformation.