| Background:Tumor necrosis factor α inducible protein family(TNFAIPs)is a protein family induced by tumor necrosis factor α(TNF-α).The members of TNFAIPs cause the occurrence and development of a variety of diseases by participating in cell apoptosis,differentiation,immune and inflammatory response,signal transduction and other processes,especially in malignant tumors and autoimmune diseases.Tumor necrosis factor-α-inducible protein 2(TNFAIP2)is an evolutionarily conserved single-copy gene that is induced by TNF-α in human umbilical cord vascular endothelial cells in 1992.TNFAIP2 protein is mainly localized in the cytoplasm and Golgi apparatus,and is also expressed in the nucleus,nuclear membrane,and membrane folds and processes of actin.TNFAIP2 is involved in inflammatory response,angiogenesis,cell proliferation,adhesion,metastasis,invasion,tunneling nanotubes formation and other functions.Previous studies have shown that TNFAIP2 is expressed in a variety of tumors,such as nasopharyngeal carcinoma,breast cancer,non-small cell lung cancer,esophageal cancer.TNFAIP2 has been confirmed to be involved in the metastasis and invasion of nasopharyngeal carcinoma,breast cancer,esophageal cancer and other tumors,and has the potential as a biomarker for tumor diagnosis and prognosis.Our previous study found that TNFAIP2 gene polymorphisms(SNPS)were associated with the risk of gastric cancer,and serum TNFAIP2 protein expression was significantly higher in gastric cancer patients than in healthy subjects.However,the pan-cancer expression characteristics of TNFAIP2 and its role and mechanism in gastrointestinal cancer are still unclear.In summary,this study analyzed TNFAIP2 expression and survival data of 33 tumors in The Cancer Genome Atlas(TCGA)by bioinformatics methods.The expression panorama of TNFAIP2 in pan-cancer and its prognostic potential were revealed from multiple dimensions such as mRNA level,DNA level and epigenetics.On this basis,we further focused on gastrointestinal cancer,and explored the expression difference of TNFAIP2 in gastrointestinal diseases and its correlation with the risk/prognosis of gastrointestinal cancer.At the same time,the effect and mechanism of TNFAIP2 expression on the biological behavior of gastrointestinal cancer cells were further explored by in vitro experiments,in order to clarify whether TNFAIP2 can be used as a target for the diagnosis and prognosis evaluation of gastrointestinal cancer,and to provide scientific basis for the diagnosis and individualized precision treatment of gastrointestinal cancer.Objectives:To clarify the expression characteristics of TNFAIP2 in pan-cancer,explore the relationship between TNFAIP2 expression and risk/prognosis of gastrointestinal cancer,and elucidate the influence and mechanism of TNFAIP2 on biological behavior of gastrointestinal cancer cells.Methods:Part Ⅰ: TNFAIP2 pan-cancer expression profile construction1.Download TNFAIP2 expression data of 33 tumors in TCGAThe RNA-seq expression,clinical information(survival status,clinical stage,survival time),tumor mutation burden(TMB),microsatellite instability(MSI),tumor micro-environment(TME),immune cell infiltration,gene mutation,copy number variant(CNV),DNA methylation data of tumor tissue samples(10363 cases)and paracancerous tissue samples(666 cases)in 33 tumor types were downloaded from the TCGA database using UCSC XENA(https://xenabrowser.net/).2.TNFAIP2 pan-cancer expression profile construction2.1 Pan-cancer analysis of TNFAIP2 expression characteristicThe differential expression of TNFAIP2 is identified by Deseq2 software package in R(v4.2.2).| log FC | ≥2 and adjusted P values<0.05 were identified as a differential expression in each tumor.2.2 The correlation analysis between TNFAIP2 expression and clinicopathological parameters/prognosisThe correlation between TNFAIP2 expression and clinical stage was analyzed using student’s t-test.Kaplan-Meier curves and forest plots were used to describe the association between TNFAIP2 expression and prognosis in 33 tumor types.P < 0.05 was considered statistically significant.Prognostic analyses included: overall survival(OS),disease-specific survival(DSS),progression free interval(PFI)and disease free interval(DFI).2.3 Correlation between TNFAIP2 expression and tumor molecular phenotypic characteristics2.3.1 The correlation analysis between TNFAIP2 expression and TMB/MSITMB is defined as the number of somatic cells,coding,base substitution and Nedel mutations in Megabytes per genome.The MSI score is derived from the TCGA database.R(v4.2.2)was used to calculate TMB and MSI of each sample in 33 different tumors,and the correlation between TNFAIP2 expression and TMB and MSI was analyzed by Spearman test.2.3.2 The correlation analysis between TNFAIP2 expression and tumor immune microenvironmentThe contents of stromal cells and immune cells in tumor microenvironment of 33 tumor types were calculated to compare the correlation between TNFAIP2 expression and the number of stromal cells and immune cells and analyze the correlation between TNFAIP2 expression and different immune cells.We screened 55 immune-related genes to explore the co-expression of immune-related genes and TNFAIP2.2.3.3 The analysis of TNFAIP2 gene mutation,CNV,DNA methylation characteristicsTNFAIP2 gene mutation,CNV and DNA methylation data in TCGA database were analyzed.The mutation frequency was calculated by the number of gene mutations.The frequency of CNV in each tumor type was calculated by the ratio of CNV amplification to CNV deletion.Differential expression of TNFAIP2 DNA methylation was identified by the Limma package of R(v4.2.2).The correlation between TNFAIP2 expression and gene mutation,CNV,DNA methylation and prognosis was analyzed.3.TNFAIP2 GSEA enrichment analysisThe different tumor KEGG gene set were download from GSEA web site(https://www.gseamsigdb.org/gsea/downloads.jsp).The TNFAIP2 expression matrix was analyzed by R(v4.2.2)and the differentially expressed genes between TNFAIP2 high expression group and low expression group were compared.The enrichment scores were calculated according to the degree of difference and enrichment to analyze the signaling pathways involved in TNFAIP2 in different tumors.An FDR < 0.05 was considered statistically significant.Part Ⅱ: Association of TNFAIP2 expression with risk / prognosis of gastrointestinal cancer1.Subjects: From October 2012 to June 2019,at the First Affiliated Hospital of China Medical University,pathological sections and fresh tissue samples were collected from patients diagnosed with non-atrophic gastritis,atrophic gastritis,gastric cancer,and patients diagnosed with anal inflammatory diseases,adenoma,and colorectal cancer.There were 39 cases of non-atrophic gastritis,101 cases of atrophic gastritis,256 cases of gastric cancer,54 cases of anal inflammatory disease,194 cases of adenoma and 134 cases of intestinal cancer.The fresh tissue samples included 21 pairs of gastric cancer(gastric cancer and adjacent normal tissues)and 60 pairs of colorectal cancer(colorectal cancer and adjacent normal tissues).At the same time,the clinicopathological data and survival information of patients with gastrointestinal cancer were collected and the survival of patients with gastrointestinal cancer was followed up every six month.2.Methods: Total RNA was extracted by Trizol method,and the expression level of TNFAIP2 mRNA in gastrointestinal tissues was detected by Real-time quantitative PCR(RT-qPCR).The expression of TNFAIP2 protein in gastrointestinal tissues was detected by immunohistochemical staining.3.SPSS(v26.0)was used for data processing and Graph Pad(v8.0)was used for analysis and mapping.2-ΔΔct was performed to calculate the relative RNA expression.The differences of measurement data between groups were evaluated by t test or nonparametric test.ROC curve was used to analyze the diagnostic efficacy.The correlation between TNFAIP2 expression and clinicopathological parameters was analyzed by χ~2 test or Fisher exact test.Kaplan-Meier analysis and univariate/multivariate COX analysis were used to evaluate the correlation between TNFAIP2 expression and the prognosis of gastrointestinal cancer.P < 0.05 was considered statistically significant.Part Ⅲ: The effect of TNFAIP2 expression on biological behavior of gastrointestinal cancer cells and its mechanism1.Subjects: Gastric cancer cell line AGS and colorectal cancer cell line SW620 were purchased from the national experimental Cell resource sharing platform and both cells had STR identification certificates.2.TNFAIP2 overexpression plasmid was constructed and transfected into gastrointestinal cancer cells to construct TNFAIP2 overexpression gastrointestinal cancer tool cell lines.3.Total RNA was extracted by Trizol method,and the expression level of TNFAIP2 mRNA in gastrointestinal cancer cells was detected by RT-qPCR.Western Blot was used to detect the protein expression level of TNFAIP2.4.CCK-8 cell activity assay and EdU fluorescence staining were used to evaluate the effect of TNFAIP2 on the activity and proliferation of gastrointestinal cancer cells.5.Hoechst staining and Western Blot detecting the apoptosis-related proteins expression were used to to evaluate the effect of TNFAIP2 on the apoptosis of gastrointestinal cancer cells.6.Cell wound healing assay,transwell migration/invasion assay were used to evaluate the effect of TNFAIP2 on the migration and invasion of gastrointestinal cancer cells.7.Western Blot detecting the expression of Epithelial-Mesenchymal Transition(EMT)-related proteins was used to evaluating the effect of TNFAIP2 on EMT in gastrointestinal cancer cells.8.Bioinformatics analysis was used to identify the TNFAIP2-enriched signaling pathway in gastrointestinal cancer tissues.Western Blot was used to detect the expression changes of key proteins in the pathway after overexpression of TNFAIP2,and the biological behavior of gastrointestinal cancer cells was observed at the same time to clarify the mechanism of TNFAIP2 affecting the biological behavior of gastrointestinal cancer cells.Results:Part Ⅰ: TNFAIP2 pan-cancer expression profile construction1.Pan-cancer analysis of TNFAIP2 expression characteristic1.1 TNFAIP2 expression characteristics in different tumorsTNFAIP2 expression was differentially expressed in 13 kinds of cancers among 33 type of tumors.In STAD,BLAD,BRCA,CHOL,LUAD,LUSC,THCA,ESCA,KIRP,UCEC,LIHC and KIRC,TNFAIP2 expression in tumor tissues was higher than that in adjacent tissues.In KICH,the expression of TNFAIP2 in tumor tissues was lower than that in adjacent tissues.1.2 Correlation between TNFAIP2 expression and clinical parameters and prognosisIn BLCA,BRCA,SKCM,KICH,PAAD,KIRP,THCA,LUSC and TGCT,TNFAIP2 expression was correlated with tumor stage.In STAD,BLCA and SKCM,the TNFAIP2 high expression group had a longer OS.In GBM,KIRC,THYM,LGG and LAML,the TNFAIP2 low expression group has an OS advantage.1.3 Correlation between TNFAIP2 expression and tumor molecular phenotypic characteristics1.3.1 Correlation of TNFAIP2 expression with TMB and MSITNFAIP2 expression was positively correlated with TMB expression in STAD,COAD,THYM,THCA,KIRP and SARC.TNFAIP2 expression was negatively correlated with TMB in 7 kinds of tumors,including LUSC,SKCM,PAAD,LIHC,LAML,GBM and CHOL.TNFAIP2 expression was positively correlated with MSI in 5tumors,including colon cancer,breast cancer,lung squamous cell carcinoma,lung adenocarcinoma and thyroid cancer.TNFAIP2 expression was negatively correlated with MSI in four tumors,including PAAD,UCS,TGCT and PCPG.1.3.2 Correlation between TNFAIP2 expression and tumor immune microenvironmentIn COAD,READ,DLBC,GBM,KICH,KIRC,LAML,LGG,LIHC,LUAD,PCPG,PRAD,SARC,SKCM,TGCT,THCA andUVM,the higher stromal and immune cell content,the higher TNFAIP2 expression.In STAD,ACC,BRCA,LUSC and OV,the higher content of immune cells,the higher expression of TNFAIP2.In BLCA,the higher stromal cells and immune cells content,the lower expression of TNFAIP2.In THYM,the higher stromal cell content,the higher TNFAIP2 expression.The immune cells associated with TNFAIP2 expression were M2 macrophages,M1 macrophages,regulatory T cells and CD8+ T cells.The immune-related genes co-expressed by TNFAIP2 included TNFRSF9,TNFRSF4,LGAL69,HAVCR2 and LAIR1.1.3.3 TNFAIP2 gene mutation,CNV,and DNA methylation characteristicsTNFAIP2 gene mutations are found in 18 types of cancers,including UCEC,COAD,SKCM,STAD,CESC,LUAD,LUSC,BRCA,GBM,OV,ACC,BLCA,DLBC,ESCA,LGG,PAAD,READ and THCA.TNFAIP2 has the highest mutation rate in COAD,STAD andUCEC,but it has no significant correlation with prognosis.TNFAIP2 copy number variations were detected in all 33 tumors.The highest proportion of copy number amplification was found in TGCT,KICH,and HNSC,while the highest proportion of copy number loss was found in CHOL,MESO and CESC.In STAD,COAD and READ,the proportion of copy number loss is higher than that of copy number gain.TNFAIP2 copy number variations were associated with patient survival in four tumors,including KIRP,LGG,MESO andUCEC.TNFAIP2 DNA methylation was differentially expressed in seven tumors,including COAD,READ,LIHC,HNSC,KIRC,BLCA and LUSC.TNFAIP2 DNA methylation is significantly increased in stage III-IV BLCA,BRCA,TGCT and LIHC.In BLCA,LGG,THYN andUCEC,TNFAIP2 DNA methylation level in age ≤60 years group was higher than that in age > 60 years group.In STAD,READ,SARC,ESCA,GBM,KIRC,KIRP,DLBC,MESO,BLCA andUVM,high TNFAIP2 DNA methylation was associated with favorable prognosis.In BRCA,CHOL,LGG,PAAD,PCPG,THYN andUCEC,high TNFAIP2 DNA methylation indicates poor prognosis.2.TNFAIP2 GSEA enrichment analysisTNFAIP2 regulates multiple immune-related signaling pathways,including:chemokine signaling pathway,cytokine-cytokine receptor pathway,T cell receptor signaling pathway,JAK-STAT signaling pathway and NK cell-mediated cytotoxic signaling pathway.Part Ⅱ: Association between TNFAIP2 expression and risk/prognosis of gastrointestinal cancer1.TNFAIP2 expression profile in different gastrointestinal diseasesThe expression of TNFAIP2 mRNA in gastric cancer tissues was higher than that in adjacent normal tissues(P=0.013).There was no significant difference in TNFAIP2 mRNA expression between colorectal cancer tissues and adjacent normal tissues(P=0.21).With the malignant evolution of non-atrophic gastritis,atrophic gastritis,and gastric cancer,the expression of TNFAIP2 gradually increases(non-atrophic gastritis vs.atrophic gastritis,P < 0.001;atrophic gastritis vs.gastric cancer,P < 0.001;nonatrophic gastritis vs.gastric cancer,P < 0.001).Similarly,TNFAIP2 protein expression was gradually increased with the progression of anal inflammatory disease,adenoma,and colorectal cancer(anal inflammatory disease vs.adenoma,P < 0.001;adenoma vs.colorectal cancer,P < 0.001;anal inflammatory disease vs.colorectal cancer,P < 0.001).2.Evaluation of TNFAIP2 expression for the diagnostic efficacy of gastrointestinal cancerROC curve analysis showed that TNFAIP2 expression had diagnostic value for gastric cancer(AUC=0.8039,P<0.0001).TNFAIP2 expression also had diagnostic value for colorectal cancer(AUC=0.8345,P<0.0001).3.Correlation between TNFAIP2 expression and clinicopathological parameters of gastrointestinal cancerIn gastric cancer tissues,TNFAIP2 expression was significantly correlated with three clinicopathological parameters,including growth pattern(P=0.006),nerve invasion(P=0.024)and Ki-67 expression level(P=0.014).TNFAIP2 low expression was more common in gastric cancer with invasive growth,positive neural invasion and Ki-67 expression > 70%.In colorectal cancer,TNFAIP2 expression was significantly correlated with neural invasion(P=0.006)and the positive rate of neural invasion was higher in TNFAIP2 high expression group.4.Correlation between TNFAIP2 expression and prognosis of gastrointestinal cancerIn gastric cancer tissues,OS of high TNFAIP2 expression group was significantly longer than that of low expression group(median OS: not reached vs.50 months,P=0.038).Colorectal cancer tissues with high TNFAIP2 expression had a significantly lower OS than those with low TNFAIP2 expression(median OS: 107 months vs.not reached,P=0.044).Univariate COX analysis showed that TNFAIP2 was a favorable prognostic factor for gastric cancer(P=0.043;HR=0.501,95%CI =0.257-0.978).The incidence of adverse prognostic events in the TNFAIP2 low expression group was twice as high as that in the TNFAIP2 high expression group.However,multivariate COX analysis showed that TNFAIP2 expression was not an independent prognostic factor for gastric cancer(P=0.504;HR=0.792,95%CI =0.400-1.568).TNFAIP2 expression was a poor prognostic factor in colorectal cancer(P=0.049;HR=1.989,95%CI =1.004-3.938)and were independent prognostic factors for colorectal cancer(P=0.015;HR=2.399,95%CI =1.189-4.840).Part Ⅲ: The effect of TNFAIP2 expression on biological behavior of gastrointestinal cancer cells and its mechanism1.The construction of TNFAIP2 overexpression tool cellsThe transfection efficiency was up to 70% in gastric cancer AGS and colorectal cancer SW620 cell lines.In AGS cell line,the overexpression efficiency was 100-150times(P < 0.01).In SW620 cell line,the overexpression efficiency was 200-250 times(P< 0.001).2.Effects of TNFAIP2 on the viability and proliferation of gastrointestinal cancer cellsCCK-8 cell activity assay showed that TNFAIP2 overexpression could not affect the proliferation activity of AGS cells,but significantly enhanced the proliferation activity of SW620 cells(P < 0.01).The results of EdU fluorescence staining proliferation assay showed that the proliferation ability of AGS cells after TNFAIP2 overexpression was significantly lower than that of the control group(P< 0.001),while the proliferation ability of SW620 cells was significantly higher than that of the control group(P< 0.01).3.Effects of TNFAIP2 on the apoptotic capacity of gastrointestinal cancer cellsHoechst staining showed that the apoptosis ability of AGS cells was significantly increased after TNFAIP2 overexpression(P < 0.01).The pro-apoptotic genes Caspase3,Cleaved-Caspase3,Bax and Bad were significantly up-regulated and the anti-apoptotic gene Bcl-2 was significantly down-regulated.Overexpression of TNFAIP2 significantly reduced the apoptotic ability of SW620 cells(P < 0.001).The pro-apoptotic genes Caspase3,Cleaved-Caspase3 Bax and Bad were significantly down-regulated,while the anti-apoptotic gene Bcl-2 was significantly up-regulated.4.Effects of TNFAIP2 on migration and invasion of gastrointestinal cancer cellsWound-healing assay showed that the migration ability of AGS cells was significantly decreased after TNFAIP2 overexpression(P< 0.001),while that of SW620 cells was significantly increased(P< 0.01).Transwell migration and invasion assay showed that TNFAIP2 overexpression significantly inhibited AGS cell migration and invasion(P < 0.01).SW620 cells did not cross the transwell chamber.5.Effects of TNFAIP2 on EMT of gastrointestinal cancer cellsIn AGS cells,the expression of N-Cadherin,MMP2,Vimentin and Snail in TNFAIP2 overexpression group were significantly down-regulated,the expression of ECadherin was significantly up-regulated.After TNFAIP2 overexpression in SW620 cells,the expression of N-Cadherin,MMP2,Vimentin and Snail were significantly upregulated and the expression of E-Cadherin was significantly up-regulated.6.The mechanism of TNFAIP2 affecting the biological behavior of gastrointestinal cancer cellsKEGG analysis showed that the differentially expressed genes co-expressed with TNFAIP2 were mainly enriched in osteoclast differentiation,chemokine signaling pathway,NF-κB signaling pathway,toxoplasmosis and apoptosis signaling pathway in gastric cancer tissues.TNFAIP2 is mainly enriched in TH17 cell differentiation pathway,chemokine signaling pathway,Th1 and Th2 cell differentiation pathway,cell adhesion molecule pathway,antigen processing and presentation pathway in colorectal cancer tissues.It can be seen that TNFAIP2 is closely related to tumor immunity in colorectal cancer.GSEA enrichment analysis showed that TNFAIP2 was closely related to MAPK signaling pathway in colorectal cancer(P < 0.001,NSE=2.66).In vitro experiments confirmed that TNFAIP2 overexpression significantly down-regulated the expression of p-NF-κB and p-IKBα in AGS cells,and Transwell migration/invasion assay showed that AGS cell migration and invasion were significantly decreased(P < 0.001).The expressions of p-MEK1/2 and p-ERK1/2 in SW620 cells were significantly up-regulated,and the results of EdU fluorescence staining proliferation experiment showed that the proliferation of TNFAIP2 overexpression group was significantly higher than that of the control group(P =0.0013).Conclusion:1.In this study,a multi-dimensional pan-cancer TNFAIP2 expression profile was constructed.The panorama shows that TNFAIP2 is differentially expressed in a variety of tumors,and it is significantly correlated with tumor clinicopathological parameters,prognosis,tumor mutation burden,microsatellite instability,tumor immunity,gene mutation,copy number variation,DNA methylation and other tumor molecular characteristics.2.With the malignant evolution of different gastrointestinal diseases,TNFAIP2 expression gradually increases,and TNFAIP2 expression has the potential as an early warning marker for gastrointestinal cancer.3.High expression of TNFAIP2 in gastric cancer tissues indicates a good prognosis of patients with gastric cancer.TNFAIP2 is a protective prognostic factor for gastric cancer,but not an independent prognostic factor.High expression of TNFAIP in colorectal cancer tissues indicates poor prognosis.TNFAIP2 is a poor prognostic factor and an independent prognostic factor for colorectal cancer.4.TNFAIP2 can promote the apoptosis of gastric cancer cells,inhibit the proliferation and EMT of gastric cancer cells and inhibit the migration and invasion of gastric cancer cells by inhibiting the NF-κB pathway.5.TNFAIP2 can inhibit the apoptosis of colorectal cancer cells and promote the migration and EMT of colorectal cancer cells.It can promote the proliferation of colorectal cancer cells by activating the MAPK pathway. |
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