| Background and objective:Bladder Cancer(BCa)is one of the most common malignant tumors in urinary system,and its pathogenesis remains unknown.Studies have shown that AC AT1 is extensively involved in ketone body metabolism,moreover it is closely associated to carcinogenesis.However,the role of AC AT1 and its metabolite β-hydroxybutyric acid in bladder cancer is still unclear.This study aims to determine the prognostic value of AC AT1 in patients with bladder cancer,and to explore its biological function through vitro experiments.Methods:The high-throughput sequencing data of bladder cancer from TCGA and GEO databases were used to screen for ketone body metabolism related genes that are differentially expressed in bladder cancer.126 patients with pathologically confirmed bladder cancer were enrolled to examine the expression of AC AT1 in bladder cancer samples and the adjacent normal tissues by immunohistochemistry.According to the protein expression level of AC AT1 by immunohistochemistry,the patients were divided into AC AT1 high expression group and ACAT1 low expression group.The difference of prognosis between the two groups was analyzed by univariate and multivariate Cox regression.Human Protein Atlas(HPA)was used to verify the expression level of AC AT1 in bladder cancer.Gene Set Enrichment Analysis was used to screen for the signaling pathways enriched in the AC AT1 high expression group.Correlation analysis was used to investigate the correlation between AC AT1 expression and immune cell infiltration.Cell functional experiments and gene knock-down experiments were used to explore the biological function of AC AT1.The migration and invasion ability of cells were detected by wound healing assay and Transwell assay.The proliferation ability of cells was detected by CCK8 and colony formation assay.Prism8.0 was used for statistical analysis.A two-tailed test p-value less than 0.05 was considered statistically significant.Results:Utilizing the high-throughput sequencing data of bladder cancer from TCGA and GEO databases,we performed differentially expressed gene analysis,and found that ACAT1,a gene related to ketone body metabolism,was down-regulated in bladder cancer(P<0.001).Immunohistochemistry was performed in 126 pairs of bladder cancer specimens and the adjacent tissues,and the results showed that AC AT1 was significantly lowly expressed in bladder cancer tissues than in the adjacent tissues(3.84±2.71 vs 6.56±2.40,P<0.001).Univariate Cox regression analysis showed that AC AT1 low expression,muscular invasion and tumor diameter>2.65 cm were risk factors for bad prognosis of bladder cancer patients(P=0.010;P<0.001;P=0.002).Multivariate Cox regression analysis showed that ACAT1 low expression,muscular invasion and tumor diameter>2.65 cm were also independent prognostic factors for bladder cancer patients(P=0.005;P<0.001;P=0.020).Consistently,the protein expression of AC AT1 from the HP A database indicated that AC AT1 is highly expressed in normal bladder epithelial tissues,and lowly expressed in bladder cancer tissues.Gene set enrichment analysis showed that T cell and B cell receptor signaling pathway,immune effector and positive regulatory pathways in immune response,positive regulatory pathways of leukocyte proliferation and migration were all enriched in the AC AT1 high expression group(P<0.05).Immune cell infiltration analysis showed that ACAT1 was positively correlated with immune infiltration in bladder cancer(P<0.001).The efficiency of siRNA-mediated knockdown of ACAT1 was verified in bladder cancer cells(T24 and 5637).Knockdown of AC AT1 significantly inhibited the production of β-hydroxybutyric acid(P<0.05)and promoted cell proliferation,migration and invasion(P<0.05).Exogenous β-hydroxybutyric acid inhibited cell proliferation,migration and invasion(P<0.05)in a concentration-dependent manner.Conclusion:Low expression of ACAT1 is an independent prognostic factor for the bad prognosis of BCa patients.ACAT1 may inhibit the proliferation,invasion and metastasis of bladder cancer cells by β-hydroxybutyric acid. |
