| Objective Bladder cancer is one of the most common tumors in urinary system.The standard treatment for early-stage bladder cancer is surgical resection,and systemic chemotherapy is predominant treatment for advanced patients.The first-line chemotherapy drugs are mainly cisplatin plus gemcitabine.Statistics show that in the past 30 years,the long-term survival of patients with bladder cancer has not been significantly improved,mainly due to the emergence of drug resistance in the late stage of chemotherapy,while the mechanism of drug resistance is still unknown.There is no effective adjuvant sensitizing drug,and also a lack of new effective drugs.So,the study of molecular mechanism of chemoresistance in bladder cancer and development of new drugs are extremely urgent for the treatment of bladder cancer.In this study,we will reveal the molecular mechanism of chemoresistance in drug-resistant bladder cancer cell line through multi-omics approaches and CRISPR library screening technology.Methods(1)First,three types of chemo-resistant bladder cancer cell lines,gemcitabine-resistant,cisplatin-resistant,and gemcitabine-resistant,were constructed by gradually increasing concentrations of drugs.(2)Transcriptomic analysis of T24 cells and chemo-resistant T24 cells was performed using next-generation sequencing to find differentially expressed genes,and gene function enrichment analysis was performed.The combined analysis of transcriptome data and pharmacogenomic database was used to looked for possible chemo-sensitizers.Drug resistance genes that may be associated with clinical prognosis was found by combined analysis with TCGA database.Finally,key genes were validated through real-time polymerase chain reaction.(3)Proteomics analysis was performed on T24 cells and chemo-resistant T24 cells to find differentially expressed proteins by quantitative mass spectrometry.The integration analysis of transcriptomic and proteomic was also performed.(4)The CRISPR library screening technology and combined analysis with transcriptomic data and proteomic data were performed to obtain genes that could increase chemosensitivity of cisplatin-resistant T24 cell lines.Result(1)We successfully constructed three chemo-resistant models including gemcitabine resistant,cisplatin resistant,and gemcitabine-cisplatin resistant cells on two cells,T24 and UMUC-3,while 5637 could not be resistant to cisplatin persistently.(2)Transcriptomic data showed that there were 304,267,and 463 up-regulated genes and 138,212,and 837down-regulated genes in gemcitabine resistant,cisplatin resistant,and gemcitabine-cisplatin resistant cells compared with parental cell line.The number of co-up-regulated genes was 56 and co-down-regulated genes was 59 across the three chemo-resistant cell lines.Functional enrichment analysis revealed that differentially expressed genes were mainly related to cell adhesion,cytokine metabolism,and inflammatory signaling pathways.A part of the differentially expressed genes were shown to be associated with clinical prognosis of bladder cancer by combined analysis with the TCGA database,and some potential chemo-sensitizers were obtained through combined analysis with pharmacogenomic data.(3)Proteomic data showed that there were 215,348,and1007 up-regulated proteins and 201,388,and 797 down-regulated proteins in gemcitabine resistant,cisplatin resistant,and gemcitabine-cisplatin resistant cells compared with parental cell line.The number of co-up-regulated proteins was 69 and co-down-regulated proteins was 66 across the three chemo-resistant cell lines.Functional enrichment analysis revealed that the differentially expressed proteins were mainly related to ribosome biogenesis,hydrogen ion transmembrane transport,actin filament organization.Furthermore,differentially expressed proteins in chemo-resistant cells were found to correlated better with gene expression profiles.(4)A total of 774 genes were obtained by negative screening of CRISPR library,and knockout of these genes could increase the sensitivity of resistant cells to cisplatin.Functional enrichment analysis showed that these genes were mainly related to nucleotide excision repair,ribosomal protein complex assembly and other functions.Among them,22 genes were similarly up-regulated in the transcriptomics or proteomics of cisplatin-resistant T24 cell line.Conclusion The bladder cancer resistant cell line induced by long-term chemotherapy drugs is stable and reliable for in vitro model.Transcriptomics and proteomics can completely reveal the landscape of m RNA and protein expression in chemo-resistant bladder cancer cells.The chemoresistance-related genes are involved in multiple intracellular biological process and signaling pathways.CRISPR library screening can effectively promote the discoveries of chemoresistance-associated genes in cancer therapy. |
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