Clinicopathological Analysis Of Traditional Serrated Adenoma And The Study Of The Role And Mechanism Of MUC2 In Colorectal Cancer

Part Ⅰ:Clinicopathological analysis of 189 traditional serrated adenomasBackgroundColorectal cancer(CRC)is the most common digestive system tumor.In addition to the classic adenoma-carcinoma sequence,about 15%to 25%of CRC progress through the serrated neoplasia pathway.Traditional serrated adenoma(TSA)is a particular subtype of colorectal serrated polyps with complex structures,accounting for approximately 1%of the serrated polyps.Epidemiological evidences suggest that patients with TSA have a higher risk of advanced neoplasia than those with conventional adenomas.The typical morphological features of TSA include villiform architecture,narrow serrated gaps,ectopic crypt foci,intense cytoplasmic eosinophilia,and rod-shaped nuclei.Endoscopically,TSA appears as reddish,protruded or pedunculated lesions in white light and "pinecone-like" or"branch coral-like" lesions in macroscopic observation.However,TSA has a papillary structure similar to villous adenoma,resulting in a low detection rate of TSA.Molecular analysis showed that most TSA were caused by KRAS or BRAF mutations,and a few had Microsatellite instability(MSI)caused by MLH1 methylation.However,molecular markers lack specificity and sensitivity,and molecular detection has limitations in the diagnosis of TSA.Therefore,it is of great significance to strengthen the understanding of the clinicopathological characteristics of TSA malignant transformation for the prevention of CRC.Objectives1.To analyze the clinicopathological baseline characteristics of 189 cases of TSA,to explore the clinicopathological characteristics and related risk factors of malignant transformation of TSA,and to provide data support and theoretical basis for the diagnosis and prognosis evaluation of TSA.2.Immunohistochemistry(IHC)of MUC2,MUC5AC,MUC6 and mismatch repair(MMR)proteins was performed to investigate the expression characteristics of mucins and MMR proteins in TSA.Methods1.A total of 189 cases diagnosed by pathology of TSA were included.The relevant clinicopathological data included gender,age,tumor size,growth location,precursor lesion,growth pattern,etc.The correlation between gross morphology of TSA and lesion location was analyzed.2.According to the presence or absence of malignant tumor components,the TSA was divided into the ordinary group and the TSA with cancer group.The differences between the two groups were analyzed and the risk factors of malignant transformation of TSA were explored.3.The TSA with cancer group was further divided into intramucosal group and submucosal group according to whether the tumor invaded the muscularis mucosa,and the risk factors related to tumor progression in malignant TSA were explored.4.The expression of MUC2,MUC5AC,MUC6 and MMR proteins in 189 cases of TSA was detected by IHC and the results were statistically analyzed.Results1.Logistic multivariate regression analysis showed that the risk factors of TSA with cancer group were larger diameter(OR=1.543,95%CI=1.159-2.055,P=0.003),tumor located in the left colon and rectum(OR=3.015,95%CI=1.097-8.286,P=0.032),sessile protruding growth pattern(OR=6.600,95%CI=2.346-18.568,P<0.001).TSA derived from sessile serrated lesion was not prone to malignant transformation(OR=0.270,95%CI=0.095-0.766,P=0.014).Correlation analysis showed that the flat TSA was mostly located in the right colon.Its distribution was statistically different from pedicled polyps,sessile protrusions and lateral spreading TSA.2.ROC curve analysis showed that the area under the curve(AUC)of lesion size for differentiating malignant TSA was 0.774(95%CI=0.705-0.843,P<0.001),the best cut-off value was 1.65cm,the sensitivity was 85.7%,and the specificity was 58.6%.3.Logistic multivariate regression analysis showed that poor differentiation(OR=35.946,95%CI=3.318-389.439,P=0.003)and smoking(OR=63.266,95%CI=1.371-2918.379,P=0.034)were risk factors for tumor progression in malignant TSA.4.IHC results showed that one case in the TSA with cancer group had MLH1 and PMS2 synchronous deletion,and one case in the normal group only had PMS2 deletion.MUC5AC was highly expressed in total TSA.However,compared with the normal group,the expression of MUC5AC and MUC2 in the TSA with cancer group decreased There were significant differences in the expression of MUC2 and MUC5AC between the two groups.Conclusions1.The growth pattern of TSA was related to the location of the lesion.TSA with flat type was more likely to occur in the right colon,while TSA with protruding type was more likely to occur in the left colon.2.Larger diameter of the lesion,location in the left colon and rectum,and protruding type of the lesion are the risk factors for malignant transformation of TSA.The lesion size was an important predictor of malignant TSA,and the diagnostic cut-off value was 1.65cm.Morphologically,TSA derived from SSL is not prone to malignant transformation,so the diagnosis of the morphology around the lesion is very important.3.Deletion of MLH1 and PMS2 was observed in one case in the TSA with cancer group,suggesting that the malignant transformation of TSA may be through MSI pathway.However,this result needs to be verified by a larger sample size and further molecular detection.Down-regulation of MUC2 expression and up-regulation of MUC5AC expression are observed in the process of malignant transformation of TSA.The abnormal expression of MUC2 and MUC5AC may play an important role in the occurrence and development of CRCPart Ⅱ:The study of the role and mechanism of MUC2 in colorectal cancer BackgroundAt present,the global incidence and mortality of colorectal cancer(CRC)are still high.With the economic development and the change of dietary habits,the incidence and mortality of CRC are showing an upward trend,which brings heavy burden to the medical and health system.CRC is a malignant epithelial tumor originating from the large intestine.In addition to the classic adenoma-carcinoma sequence pathway and the serrated neoplasia pathway,the rest are caused by gene mutations.Despite the availability of effective molecular therapies,the prognosis of CRC remains poor.Meanwhile,TNM staging has limited predictive ability for prognosis of CRC patients.Therefore,it is necessary to explore new molecular markers.Mucins are the main components of mucus on the luminal surface of epithelial organs,participate in physiological processes such as epithelial cell protection,signal transduction and tissue homeostasis.The expression of mucin is tissue-specific,and abnormal expression is more common in malignant tumors.MUC2 is an important secretory protein of the human intestinal tract,secreted by goblet cells.Mucins play a crucial role in protecting the intestinal tract and maintaining intestinal homeostasis.The loss of MUC2 expression in CRC has been reported in several studies,and in the first part of our study,we observed the down-regulation of MUC2 expression in malignant TSA with cancer.However,the role and mechanism of MUC2 in colorectal cancer have not been systematically studied.Objectives1.To explore the expression of MUC2 in CRC and its relationship with prognosis using online bioinformatics database.2.To investigate the relationship between MUC2 expression and clinicopathological features of CRC.3.To investigate the effect of MUC2 knockdown on the proliferation,invasion and migration of CRC cells and the underlying molecular mechanisms in vitro.Methods1.Online bioinformatics databases were used to analyze the expression of MUC2 in CRC,its relationship with prognosis,immune cell infiltration in tumor microenvironment and molecular subtypes of CRC.2.Two hundred cases of CRC diagnosed by pathology were collected.Relevant clinicopathological data were collected,including age,gender,tumor location,differentiation,size,TNM stage,clinicopathological stage,and immunohistochemical expressions of MUC2,P53 and Ki67.3.CRC was divided into positive group and negative group according to MUC2 expression.Chi-square test was used to analyze the difference between the two groups.Ordinal logistic regression was used to analyze the effect of MUC2 expression on tumor T stage.4.Two colorectal cancer cell lines,SW480 and HCT116,were selected.The expression of MUC2 was knocked down by transfection of small interfering RNA(siRNA).RT-qPCR and western blot were used to verify the knockdown efficiency.CCK8 assay and EdU assay were used to detect the effect of MUC2 knockdown on the proliferation of CRC cells.Transwell assay was used to detect the effect of MUC2 knockdown on the invasion and migration of colorectal cancer cells.Western blot was used to detect the expression of TGFβ-Smad signaling pathway-related proteins.Results1.The analysis of online bioinformatics databases showed that the expression of MUC2 was significantly decreased in CRC.The high expression of MUC2 was associated with a better prognosis of patients.In terms of tumor immune infiltration,the expression of MUC2 was positively correlated with the infiltration of CD4+T cells,and the expression of MUC2 was related to the molecular subtypes of CRC.2.Chi-square test showed that the expression of MUC2 was correlated with tumor T stage(χ2=7.780,P=0.049),mucus proportion(χ2=15.405,P<0.001)and P53 mutation(χ2=7.423,P<0.001).Multivariate Ordered logistic multivariate regression analysis showed that T staging increased with the decrease of MUC2 expression(OR=0.404,95%CI=0.201-0.811,P=0.011).3.After MUC2 knockdown by siRNA,the proliferation,invasion and migration of SW480 and HCT116 cells were enhanced.Western blot analysis showed that the expressions of TGFβⅠ,TGFβⅡ,Smad2/3 and Smad4 were decreased in HCT116 cells after MUC2 knockdown.Conclusions1.Analysis of the online bioinformatics database showed that MUC2 is a potential prognostic molecular marker for CRC and is expected to become a new target for CRC therapy.2.Clinical data showed that with the decrease of MUC2 expression,the tumor T stage increased.3.MUC2 can inhibit the proliferation,invasion and migration of colorectal cancer cells,which may be regulated by TGFβ-Smad signaling pathway.