Impact Of NAFLD On Survival Outcomes After Intrahepatic Cholangiocarcinoma Resection And The Association Between SULT2B1b Expression And Tumor Invasiveness

Part ?.Impact of Non-Alcoholic Fatty Liver Disease on the Long-term Outcomes after Liver Resection for Intrahepatic CholangiocarcinomaBackground: Non-alcoholic fatty liver disease(NAFLD)has been an increasing cause of intrahepatic cholangiocarcinoma(ICC)worldwide.The current study aimed to evaluate the impact of NAFLD on the survival outcomes after surgical resection of ICC.Methods: Data on 1045 consecutive patients with ICC who underwent liver resection at the Eastern Hepatobiliary Surgery Hospital between 2003 and 2011 were analyzed.Propensity score matching(PSM)was used to adjust for differences in baseline characteristics between the NAFLD-ICC versus Non-NAFLD-ICC patients.Results: Compared with Non-NAFLD-ICC patients(n = 831),NAFLD-ICC patients(n = 214)were more likely to have overweight(64.0% vs.21.7%)and type 2 diabetes(15.9% vs.4.0%),and had a lower incidence of cirrhosis(7.0% vs.25.2%)(all P < 0.01).NAFLD-ICC patients had a higher surgical morbidity rate than Non-NAFLD-ICC patients(28.5% vs.18.5%,P=0.001).The 5-year recurrence,cancer-specific survival(CSS)and overall survival(OS)rates between the NAFLD-ICC and Non-NAFLD-ICC patients were not significantly different(88.5% vs.83.4%,P = 0.100;24.4% vs.29.3%,P = 0.200;and 20.4% vs.28.2%,P = 0.100).Among patients with I/II stage ICC based on the 8th AJCC/TNM staging system,NAFLD was associated with worse 5-year recurrence,CSS and OS than Non-NAFLD before and after PSM(before PSM: 86.1% vs.80.1%,P = 0.030;23.3% vs.35.1%,P = 0.030;and 20.5% vs.33.5%,P = 0.020;after PSM: 86.3% vs.75.9%,P = 0.040;23.3% vs.38.5%,P =0.040;and 21.2% vs.35.8%,P = 0.040).On multivariate analysis in the PSM cohort,NAFLD-ICC was associated with worse recurrence,CSS and OS rates versus Non-NAFLDICC(hazard ratio: 1.56,95% confidence interval: 1.17-2.08;1.43,1.05-1.95;1.44,1.06-1.95).Conclusions: NAFLD was associated with worse survival outcomes compared with NonNAFLD among patients with stage I/II ICC who underwent liver resection.Part ?.High expression of SULT2B1 b gene in NAFLD-ICC and validation of gene expressionBackground: Our previous studies have demonstrated that NAFLD was associated with worse survival outcomes compared with Non-NAFLD among patients with stage I/II ICC following liver resection.The current study aimed to investigate the candidate genes which may involve in the carcinogenesis of NAFLD-ICC using next-generation of transcriptome sequencing between NAFLD-ICC and Non-NAFLD-ICC tissue samples,which may have a potential in identifing the markers for molecular classification of ICC.Methods: Extract RNA from 43 pairs of ICC tissue samples and their corresponding adjacent normal liver tissues for transcriptome sequencing.The differentially expressed genes were screened through bioinformatics analyses,the GO and KEGG signaling pathways enrichment and literature search.The prognostic analysis was also performed according to the expression levels of differentially expressed genes.Based on the findings that the hydroxysteroid sulfate transferase(hydroxysteroid sulfate transferases)2B1b(SULT2B1b)gene was associated with the occurrence and development of NAFLD-ICC,as well as the survival outcomes,immunohistochemical staining was carried out on the paraffin-embedded sections of 105 ICC patients to identify the association between the SULT2B1 b protein expression level and the prognosis of NAFLD-ICC.Results: Transcriptome sequencing revealed multiple differentially expressed signaling molecules.SULT2B1 b gene was identified by analyzing gene expression level,the correlation between its expression level and prognosis,and analysis of signal pathways.The expression level of SULT2B1 b gene in the NAFLD-ICC tissues was significantly higher than that in the Non-NAFLD-ICC tissues,and also significantly higher than that in the normal liver tissues.The high-expression of SULT2B1 b gene was associated with worse long-term outcomes than low-expression.Immunohistochemical staining demonstrated that patients with high-expression of SULT2B1 b protein had a higher recurrence rate and a lower OS rate than patients with low-expression.Multivariate analysis showed that the highexpression of SULT2B1 b protein in NAFLD-ICC tissues and the late AJCC/TNM stage were independent risk factors for recurrence and OS in ICC patients.Conclusions: SULT2B1 b gene and its encoded protein were highly expressed in NAFLDICC tissues.The high-expression of SULT2B1 b gene was associated with worse survival outcomes then low-expression among surgically treated ICC patients.Part ? .Effect of SULT2B1 b on proliferation,migration and apoptosis of ICC cellsBackground: To examine the effect of SULT2B1 b on the malignant biological behaviors of ICC cells after confirming that SULT2B1 b expression level was markedly up-regulated in NAFLD-ICC tissues and closely associated with clinical outcomes.Methods: The SULT2B1 b gene was knocked out by small interference RNA(si RNA).Quantitative real-time PCR and western blot were used to detect the silencing efficiency.CCK-8 assay was used for detecting cell viability after silencing SULT2B1 b.The SULT2B1 b stably knock down ICC cells was developed by transfection of lenti-virus with sh RNA against SULT2B1 b.Subcutaneous nude mice model was constructed by injecting ICC cells with lenti-sh SULT2B1 b or lenti-vector control into the lower flank of female nude mice.Wound healing and transwell invasion were performed to evaluate the influence of SULT2B1 b on the migration and invasion ability of ICC cells.Flow cytometry analysis was performed to investigate the impact of SULT2B1 b knockdown on the cell cycle and apoptosis.Results: CCK-8 cell proliferation assay showed that the interference of SULT2B1 b expression in ICC cells could inhibit the proliferation of ICC cells.The subcutaneous tumorigenesis nude mice model of ICC confirmed that SULT2B1 b gene knockdown resulted in decreased proliferation capacity of ICC.Wound healing assay showed that SULT2B1 b knockdown could inhibit the migration of ICC cells and transwell matrigel invasion assay suggested that SULT2B1 b knockdown could significantly inhibit the invasion ability.Flow cytometry analysis showed that SULT2B1 b knockdown resulted in G2/M stage arrest of ICC QBC939,but did not affect the cell apoptosis.Conclusions: The interference of SULT2B1 b gene expression could significantly inhibit the proliferation,migration and invasion of ICC cancer cells.