The Related Research Of Multi-omics To Explore The Metabolic Reprogramming Of Bladder Cancer Stem-like Cells

Objective:TGF-β1 induces epithelial-mesenchymal transformation of bladder cancer cells and obtain tumor stem-like cell properties.Transcriptomics,quantitative proteomics and non-targeted metabolomics were established based on bladder cancer stem-like cells,and analysis was conducted to find the specific pathways and related genes involved in the metabolic reprogramming process of bladder cancer stem-like cells,so as to provide clues for the research on the mechanism of metabolic reprogramming of bladder cancer stem cells.Methods:Bladder cancer cells were induced by TGF-β1 to acquire tumor stem-like cell properties.Meanwhile,morphologic observation,RT-qPCR and flow cytometry were used to verify the effect of TGF-β1.Bladder cancer cells and bladder cancer stem-like cells were collected for the construction of cDNA library and after passing the quality inspection,the library was double-ended sequencing using high-throughput sequencing platform.The significantly different genes were screened with q<0.05 as the standard.q<0.05 was taken as the significant enrichment standard,and the software Clusterprofiler was used to enrichment analyze the differential genes.Proteins from bladder cancer cells and bladder cancer stem-like cells were collected and sequenced by TMT labeling.Differential protein screening,subcellular localization,GO enrichment analysis and KEGG enrichment analysis were performed by bioinformatics software.The metabolites of bladder cancer cells and bladder stem-like cells were collected and extracted for non-targeted metabolomic analysis using UHPLC-QTOFMS.After the pretreatment of the original data,the compounds with differences between the two groups were screened out.After the secondary mass spectrometry matching and the combination of positive and negative ions for repeated processing,the clearly matched differential metabolites were obtained.Metabolic pathway analysis was performed using MetaboAnalyst4.0 online tool.Pathways and genes involved in metabolic reprogramming of bladder cancer stem cells were screened by multi-omics analysis,and the biovalidation was carried out by TCGA and GEO database.Results:The cell morphology of bladder cancer cells was significantly changed,the expression of EMT and stem-related genes were up-regulated,and the proportion of CD44+cells was increased after 6 days treatment of TGF-β1.Transcriptome analysis revealed 1664 genes with significant differences,among which 851 genes were significantly up-regulated and 813 genes were significantly down-regulated.GO enrichment analysis revealed the enrichment of epithelial to mesenchymal transition,regulation of wound healing,regulation of lipid storage and other pathways.KEGG enrichment analysis revealed enrichment of PI3K-Akt signaling pathway,Glycolysis/Gluconeogenesis,and Hippo signaling pathway.Proteomic analysis revealed 867 significantly different proteins,of which 300 proteins were significantly increased and 567 proteins were significantly decreased.GO enrichment analysis showed significant enrichment in metabolic processes,Regulation of growth,and Locomotion pathways,while KEGG enrichment analysis showed significant enrichment in PI3K-Akt signaling pathway,Cell adhesion molecules(CAMS),and Glycolysis/Gluconeogenesis pathways.Metabolomics analysis revealed 71 metabolites with significant differences.KEGG enrichment analysis revealed significant enrichment in Steroid biosynthesis、Olfactory transduction、Purine metabolism signaling pathway and other pathways.Glycolysis/Gluconeogenesis pathway enrichment was found in both transcriptome and proteome.In this pathway,ALDH1A3,PKM2,BPGM,genes and their coding proteins showed significant differences in transcriptome and proteome,and the changes in the two groups were consistent.TCGA and GEO database data showed that ALDH1A3 expression was low in tumors and was positively correlated with prognosis.At the same time,the high expression of BPGM in tumor was inversely correlated with prognosis.Conclusion:Through multiple omics analysis,we found the metabolic reprogramming of bladder cancer stem cells may be associated with glycolysis pathway,also found that the multiple potential candidate genes in this pathway.It provides clues for the metabolic reprogramming of bladder cancer stem cells,but still needs further function experiment to find out the specific mechanism.