| Colon cancer is a common malignant tumor with high morbidity and mortality.In recent years,the incidence of colon cancer in our country increased significantly.The onset of the disease is insidious,and most of the clinical manifestations appear in the middle and late stages.Although the treatment of colon cancer has made great progress,the survival rate of colon cancer patients is still poor,which seriously threatens the health of Chinese people.Therefore,it is significant to explore novel biomarkers and therapeutic targets for colon cancer patients.Epithelial-mesenchymal transition(EMT)is a cell biological process in which epithelial cells obtain mesenchymal phenotypes after down-regulating epithelial properties through specific procedures.EMT promotes cell migration,tumor metastasis and drug resistance,which has an important impact on the prognosis of colon cancer.In addition,tumor cells could reprogram the immune microenvironment and promote the formation of suppressive immune microenvironment,which is also important for the progression of colon cancer.Previous studies have found a close relationship between EMT and immune microenvironment.Moreover,tumor cells and immune cells could activate the same cell biological processes through the same stimulus,they influence each other and jointly affect the prognosis of patients.Therefore,it is of great significance to identify EMT-immune-related prognostic biomarkers and potential therapeutic targets.In this study,CDKN2 A,CMTM8 and ILK were selected as the key genes with prognostic value.Furthermore,bioinformatics was used to analyze the influence of key genes on the EMT process and immune microenvironment.And the enrichment analysis was conducted to study the possible signal pathways.Among the 3 key genes,CDKN2 A has higher prognostic value,which is also a potential therapeutic target of colon cancer.Moreover,CDKN2 A has important regulatory effects on EMT and immune microenvironment.In addition,CDKN2A-related co-expression genes were used to establish prognostic model and nomogram.The model and the nomogram could significantly improve the accuracy of prognosis judgment.In order to further study the effects of CDKN2 A on the proliferation,migration and EMT process,cell experiments were conducted,and the possible signal pathways were discussed.Part One: Identification and verification of the EMT-immune-related prognostic biomarkers in colon cancer by bioinformatics methodObjective: This part aims to identify the EMT-immune-related prognostic biomarkers and potential therapeutic targets of colon cancer.Moreover,the potentially mechenism of influencing EMT process and immune microenvironment were analyzed.Methods: The GEPIA 2.0 database and GSE10950 dataset were used to conduct different expression analysis.The DEGs related to EMT and immune were identified by Innate DB,Import and EMTome databases.Furthermore,the key genes with prognostic value were screened out.GSE39582,GSE24511 and GSE29621 datasets were used to verify the prognostic value of key genes,respectively.Then,the expression of key genes were analyzed by GEPIA 2.0 database,GSE10950 dataset,Oncomine database and GSE166555single-cell dataset.Pathological specimens of our hospital were collected for immunohistochemical staining to verify the difference in expression at the protein level.Furthermore,the “spearman” method were used to analyze the correlation between key genes and cancer-associated fibroblast(CAFs)markers,EMT-related markers.In order to study the influence of key genes on immune microenvironment,the immune infiltration analysis was performed by “TIMER” and “CIBERSORT” method.Moreover,the correlation between key genes and immune checkpoints,immunosuppressive factors was analyzed by the “spearman” method.GO,KEGG and GSEA methods were used to do functions and pathways enrichment analyses.By establishing ce RNA networks,analyzing the genome mutations and copy number variation of the key genes to study their regulatory mechanisms.Cell Miner data and TCGA data were used for treatment sensitivity analysis.Results: CDKN2 A and CMTM8 were up-regulated in tumor tissues,and is significantly correlated with poor overall survival.While,ILK was down-regulated in tumor tissues,which is significantly associated with poor prognosis.Among them,the expression of CDKN2 A is significantly correlated with the N stage,lymphatic metastasis,AJCC and pathological stage.So,CDKN2 A has better clinical value.CDKN2 A,CMTM8 and ILK were significantly correlated with EMT-related markers and CAFs markers.CDKN2 A was positively correlated with CAFs markers,mesenchymal cell markers,and negatively correlated with epithelial markers.In addition,CDKN2 A,CMTM8 and ILK all have important regulatory effects on immune microenvironment.The “TIMER” algorithm indicated that CDKN2 A was positively correlated with M2 type macrophages.“CIBERSORT” algorithm suggested that CDKN2 A was negatively correlated with memory CD4+T cells(R=-0.16),naive CD4+T cells(R=-0.15)and positively correlated with T cells regulatory(R=0.17).Moreover,CDKN2 A was positively correlated with immune checkpoints expression and immunosuppressive factors expression.As a result,CDKN2 A could promote the EMT process and inhibitory immune microenvironment of colon cancer.CDKN2 A may involved in the regulation of EMT and immune microenvironment through TGF-β/Smad pathway.Finally,we found CDKN2 A have more effect on chemotherapy drug sensitivity and immunotherapy efficacy.Conclusions: CDKN2 A,CMTM8 and ILK are important EMT-immune-related prognostic markers.CDKN2 A could be seen as a better prognostic marker and potential therapeutic target of colon cancer.CDKN2 A may promote EMT progression and immunosuppressive microenvironment formation in colon cancer by regulating TGF-β/Smad pathway.Part Two: Establishment and validation of the prognostic signature of colon cancer based on CDKN2 A co-expression genesObjective: In this part,the prognostic risk model and nomogram of CDKN2 A related co-expression genes were established,and we further analyzed the prognostic value of them.Methods: CDKN2 A associated co-expression genes were screened by CCLE data.For further screening important prognostic factors and establishing the prognostic model,the expression and clinical data of TCGA were downloaded to do univariate COX and multivariate COX regression analyses.The prognostic value of the model was evaluated by K-M survival curve.Further univariate and multifactorial COX analysis were performed to determine whether the risk model had independent prognostic value.The predictive value of the signature and clinical indicators were analyzed by ROC curves.Subsequently,a nomogram was established to improve the convenience of prognosis evaluation,and the accuracy of the nomogram to predict prognosis was analyzed.Finally,the prognostic value of the signature was verified by GSE17537 dataset.Results: Among the 62 CDKN2 A co-expressed genes,NEK4(P=0.025),ELAC1(P=0.022),SLC25A38(P=0.001),HOXC9(P=0.006)were filtered out by univariate and multivariate COX regression analysis.The formula of the signature was: NEK4 expression level×(-0.86)+ELAC1 expression level×(-1.21)+SLC25A38 expression level × 1.49+HOXC9 expression level ×0.68.The K-M curve showed that the survival rate of high-risk group was significantly lower than that of low-risk group(P<0.001).The risk model also could be used as an independent prognostic factor of colon cancer patients.ROC curves showed that the AUC of 1-year,3-year and 5-year prognosis were0.740,0.746 and 0.795,respectively.The combined AUC of the signature and other clinical indicators were 0.790,0.774 and 0.809,respectively.By GSE17537 data,we found that the high risk group had a worse prognosis than the low risk group(P=0.053).Its role as an independent prognostic factor was verified by independent prognostic analysis.The AUC for 1-year,3-year,and5-year prognosis predicted by the signature was 0.899,0.668,and 0.639,respectively.The combined AUC were 0.863,0.837 and 0.827,respectively.The nomogram was a good guide for clinical prognosis assessment.Conclusions: The signature established by CDKN2 A co-expressed genes has higher prognostic value than a single index.Moreover,the nomogram could be more accurate and convenient to guide clinical prognosis assessment.Part Three: CDKN2 A could mediate the proliferation,migration and EMT process of colon cancer cells via TGF-β/Smad signaling pathwayObjective: The purpose of this part was to further verify the effect of CDKN2 A on the proliferation,migration,EMT process of colon cancer cells and the possible mechanism through in vitro cell experiments.Methods: Knockdown and overexpression of CDKN2 A in HCT116 and SW480 were performed by small interfering RNA(si RNA)transfection and overexpressed lentivirus transfection.The knockdown and overexpression effects of CDKN2 A were verified by western blot.The relative expression level of PCNA and the CCK-8 assay were used to analyze the effect of CDKN2 A on the proliferation of colon cancer cells.The effect of CDKN2 A on migration was analyzed by the wound healing assay.Then western blot assay was used to analyze the expression changes of ZO-1,E-Cadherin,Vimentin,N-Cadherin and p-Smad/Smad ratio after CDKN2 A knockdown and overexpression to determine the effect on the EMT process and the TGF-β/Smad signal pathway.Results: PCNA was down-regulated when CDKN2 A knockdown and it was up-regulated when CDKN2 A overexpression.In addition,CCK-8experiment found that CDKN2 A knockdown will result in decreased cell proliferation,while CDKN2 A overexpression promoted cell proliferation.The wound healing assay of SW480 cells showed that CDKN2 A overexpression group had higher cell migration rate.In addition,CDKN2 A could promote the up-regulation of Vimentin and N-Cadherin expression in HCT116 and SW480 cells,while down-regulation of ZO-1 and E-Cadherin expression.Thus,CDKN2 A promote the progression of EMT.We also found the decrease of p-Smad2/Smad ratio in CDKN2 A knockdown group and the increase in CDKN2 A overexpression group,which suggested that CDKN2 A promote Smad2 phosphorylation activation and activate TGF-β/Smad pathway.Conclusions: CDKN2 A could promote the proliferation,migration and EMT progression of colon cancer cells by regulating the TGF-β/Smad pathway. |
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