HMGCS2 Mediates Ketone Production And Inhibits The Proliferation And Metastasis Of Bladder Cancer And The Underlying Mechanism

Part Ⅰ Identifying the molecular markers related to bladder cancer progression using the Weighted Gene Coexpression Network Analysis(WGCNA)Objective To analyze the correlation between gene expression profiles of bladder cancer patients and clinical data using weighted gene co-expression network analysis.Methods The bladder cancer expression microarray was obtained from the NCBI Gene Expression Omnibus(GEO)database and TCGA database.The R package limma was used to screen the differentially expressed genes(DEGs)between the MIBC and the NMIBC sample.The DEGs were classified into different modules.Pearson’s correlation was used to calculate the correlation coefficient between different modules and clinical features,and to identify the clinically most related modules.The hub genes were identified from the clininically related module according to gene significance and module membership.Unpaired t-test was used to detect the difference of hub gene expression between different clinical features.One-way ANOVA was used to analyze the difference of hub genes expression across different tumor stages.The bladder cancer tissue microarray was used to detect the protein level of hub genes.Results 873 DEGs were screened with FDR <0.05 and |Fold Change| ≥ 1.2.A total of6 co-expression modules were identified.Pearson’s correlation analysis showed that the yellow module(hub module)was associated with pathological stage and invasion and had the highest correlation(cor = 0.5,p = 4e-07;cor = 0.53,p = 4e-06).A total of 19 hub genes were identified,which were further verified by survival analysis,ROC curve analysis and q PCR of bladder cancer tissues,and finally determined the real hub genes related to the stage and prognosis of bladder cancer.Furthermore,the expression of HMGCS2 protein was detected using bladder cancer tissue microarray.It was finally determined that HMGCS2 was significantly down-regulated in bladder cancer tissues,and the expression level decreased as the tumor stage increased.Conclusion HMGCS2 may be used as a biomarker to identify different pathological stages of bladder cancer to predict the tumor progression of bladder cancer.This molecular marker may be important for improving risk stratification,treatment decision and prognosis prediction of bladder cancer patients.Part Ⅱ The study of the effect of HMGCS2 on ketone bodies production and the biological behavior of bladder cancer cellsObjective To investigate the effect of HMGCS2 on the proliferation and migration of bladder cancer cells in vitro and in vivo.Methods: q PCR and western blot were used to detect the expression of HMGCS2.Transient cell transfection was used to establish a HMGCS2 overexpression cell model.Lentivirus sh RNAs was used to construct stable HMGCS2 overexpression and knockdown models of bladder cancer cells.MTT assay and clone formation assay were used to detect cell proliferation and cloning ability of bladder cancer cells.Transwell assay and wounding heal assay were used to detect cell migration ability.Western blot and immunofluorescence were used to detect the protein levels.β-hydroxybute assay was used to detect the level of β-hydroxybutyte.Xenograft experiment was used to detect the growth and tumorigenic ability of tumor cells in vivo.Results qPRC and western blot results showed that HMGCS2 m RNA and protein levels were significantly reduced in bladder cancer cells.Western blot detected the expression of HMGCS2 in overexpression and knockdown cell line models,the results showed bladder cancer cells T24 and 5637 had significantly increased protein levels of HMGCS2.After using different sh RNA to knockdow HMGCS2 m RNA expression,their protein level of HMGCS2 were significantly reduced.β-hydroxybutyte assay showed that β-hydroxybutyte was significantly increased after HMGCS2 overexpression and significantly reduced after HMGCS2 knockdown.MTT assay results showed β-hydroxybutyte inhibited the proliferation of bladder cancer T24 cells.MTT assay and clone formation assay showed that the proliferation and cloning ability of cells were decreased in HMGCS2 overexpression cells and were significantly increased in HMGCS2 knockdown cells.Xenograft assay showed that knockdown of HMGCS2 significantly promoted tumor growth in vivo.q PCR,Western blot and immunofluorescence experiments showed that the levels of E-cadherin increased in T24 and 5637 HMGCS2 overexpressing cells,and that N-cadherin and vimentin were significantly increased in HMGCS2 knockdown cells.Ketone body supplementation rescued the proliferation and migration ability of cells after HMGCS2 knockdown.Conclusion HMGCS2 inhibited the proliferation and migration of bladder cancer cells by promoting the ketongenesis of bladder cancer cells,and inhibited the EMT of bladder cancer cells.Part Ⅲ The mechanism of HMGCS2 promoting ketone bodies production and inhibiting the proliferation and migration of bladder cancer cells Objective To explore the mechanism of HMGCS2 gene regulating the proliferation,migration and EMT of bladder cancer cells.Methods Seahorse assay was used to detect glycolysis changes in HMGCS2 overexpressing bladder cancer cells.RNA-sequencing was used to detect changes of genes and signaling pathways in HMGCS2 overexpressing bladder cancer cells.q PCR was used to verify RNA-sequencing results.Western blot was used to detect the effect of ketone bodies on histone H4 β-hydroxybutyration.Western blot was used to detect the histone H4 β-hydroxybutyration in the HMGCS2 overexpression cells.Chromatin immunoprecipitation(Chip)assay was used to detect the binding between histone H4 and the promoter region of downstream genes.Results The results of the Seahorse glycolysis assay showed that after overexpression of HMGCS2 the extracellular acidification rate(ECAR)was significantly reduced,and the cells glycolytic capacity was reduced in bladder cancer T24 and 5637 cells.Bladder cancer cell RNA-sequencing results showed that upregulated genes in HMGCS2 overexpression are significantly enriched in cell cycle,DNA replication and cell division,and downregulated genes enriched in the cellular response to chemical stimulus,cell mobility and cell migration.Histone β-hydroxybutyration levels were significantly increased in bladder cancer liver cancer cells after the treatment of β-hydroxybute.We also found that histone β-hydroxybutyration was significantly increased after HMGCS2 overexression and decreased after HMGCS2 knockdown in bladder cells.Chip assay showed that the histone H4 β-hydroxybutyration had increased binding to promoter region of genes such as CXCL3,CXCL6,JAM2,FN1,ASTN1 and MMP10 and inhibits their expression in HMGCS2 overexpressed bladder cancer cells.Conclusion HMCGS2 inhibited the production of ATP by inhibiting the glycolysis process of bladder cancer cells.HMGCS2 promoted histone H4 β-hydroxybutyration by increasing ketone bodies production,and inhibited the expression of genes related to cell growth and migration such as CXCL3,CXCL6 and MMP10 by increasing the binding of promoter region of these genes,thereby inhibiting the proliferation and migration of bladder cancer cells.