Exploration Of Prognostic Value Of SIRT2 And Development Of A Survival Prediction Model For Patients With Resectable Cholangiocarcinoma

Objective:To explore the expression,potential mechanism and clinical significance of silence information regulator 2(SIRT2)in cholangiocarcinoma(CCA),and a survival prediction model for patients with resectable CCA was also developed.Methods:Firstly,the RNA sequence data of CCA tissues and normal tissues were analyzed based on The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)databases to compare the expression level of SIRT2 m RNA in CCA tissues and corresponding paired normal tissues.In order to explore the potential mechanism of SIRT2 activity in CCA,the STRING database was used to construct a protein interaction network for physical binding of SIRT2.The Linkedomics database was used to explore SIRT2 co-expression genes and to perform functional enrichment analysis of co-expression genes.The R package "DESeq2" was used to screen differentially expressed genes(DEGs)in the high and low SIRT2 expression groups,with |log FC|>1.5 and adjusted P<0.05 as screening criteria.The Metascape database was used for DEGs enrichment analysis,and the R package "cluster Profiler" was used for gene set enrichment analysis(GSEA),and FDR<0.25,adjusted P<0.05 and standardized enrichment score |NES|>1 were considered significantly enriched.Subsequently,a total of 89 patients with CCA who underwent surgery and confirmed by pathology in the First Affiliated Hospital of Bengbu Medical College from January 1,2016 to December 31,2021 were retrospectively collected,including which 44 cases of adjacent non-tumor(negative surgical margin)(ANT)bile duct tissue samples was concurrently collected.Immunohistochemical(IHC)staining was5performed to detect the expression of SIRT2 in the above CCA tissues and ANT tissues.The clinicopathological characteristics of the 89 CCA patients were collected from inpatient medical records.These patients were followed up by telephone and the overall survival was calculated.The association between SIRT2 expression and clinicopathological characteristics of CCA patients was analyzed based on this investigator cohort.The Kaplan-Meier survival analysis was used to compare the differences in OS between SIRT2-low and SIRT2-high CCA patients,and COX regression analysis was conducted to determine the independent predictors of OS in CCA patients.Finally,a survival prediction model for patients with resectable CCA was constructed based on the COX regression results,the calibration curve and the time-dependent receiver operating characteristic curve(ROC)were used to evaluate the model,and the predictive power between this model and the AJCC/TNM staging system(8th Edition)was ultimately compared.Results: Analysis of transcriptomic data based on the TCGA database revealed that SIRT2 m RNA level was significantly higher in CCA(CHOL)tissues compared to normal tissues(P<0.001),and SIRT2 m RNA expression level in CCA tissues was also showed to be significantly higher than that in their corresponding paired normal bile duct tissues(P<0.05).GSE107943 dataset(platform number GPL18573)in GEO database further validated the over-expression of SIRT2 m RNA in CCA tissues and the differential analysis results between paired samples.Analyses from the STRING database identified 40 proteins that physically interact with SIRT2,including SFN,XRCC6,CBX5.Through Linkedomics database analyses TOP50 genes that were positively and negatively correlated with SIRT2 m RNA expression,were obtained respectively.The positively correlated genes included ATG4 A,SMPD1,NFKBIB,etc,and the negatively correlated genes include ZNF624,SMARCC1,ACVR2 B,etc.The function enrichment results of Metascape database suggested that DEGs were mainly enriched in hormone level regulation,intercellular adhesion,Notch signaling pathway regulation,and other functions.The results of GSEA implicated that the differential genes between patients with high and low SIRT2 expression were significantly enriched in ribosomes,peroxisome,PPAR signaling pathways,and in various metabolic related pathways such as oxidative phosphorylation,amino acid metabolism,and fatty acid metabolism.Based on the analyses of researchers cohort,SIRT2 IHC scores were found to be significantly higher in CCA tissues compared to ANT tissues(P<0.01),and the results were also true from comparison between CCA and their paired ANT samples(P<0.01).Clinicopathological characteristics analysis revealed that SIRT2 expression was related to blood triglycerides(P=0.024),tumor size(P=0.035),and lymph node metastasis(P=0.021).Survival analysis showed that patients with high SIRT2 expression had shorter OS than those with low SIRT2 expression(21 months vs.29 months,P=0.015).Univariate COX regression analysis suggested that pathological differentiation(P=0.010),TNM stage(P=0.002),postoperative treatment(P=0.040)and SIRT2 expression(P=0.016)were associated with the prognosis of postoperative CCA patients.Multivariate COX regression analysis further revealed that TNM stage(HR: 2.100,95%CI: 1.129-3.904,P=0.019)and SIRT2 expression level(HR: 1.726,95%CI: 1.012-2.944,P=0.045)were independent predictors of OS in postoperative CCA patients.Finally,a nomogram based on SIRT2 and meaningful clinicopathological parameters for prediction of survival for postoperative CCA patients was constructed.The C-index of the model was 0.675,and the area under the time-dependent ROC curve for the first,second,and third years was 0.879,0.778,and 0.953,respectively.Compared to the area under the time-dependent ROC curve for the first,second,and third years for the AJCC/TNM staging system(8th Edition)as 0.745,0.621,and 0.612,this model showed a better predictive effect.Conclusion: SIRT2 is highly expressed in CCA tissues and associated with poor prognosis in patients with resectable CCA.The nomogram based on SIRT2 for prediction of survival of postoperative CCA patients is identified to have more effective predictive power than the AJCC/TNM staging system(8th Edition).