| Objective:Urinary bladder neoplasm is one of the most common cancers worldwide.Up to 70%of patients with bladder tumors are initially diagnosed as non-invasive bladder tumors,and more than 70%of them may recur in the future or even relapse multiple times.Although the 5-year survival rate of bladder tumor patients is higher than those of other cancer patients,the recurrence rate is still extremely high,which aggravates the patient's mental and economic burden.Therefore,it is urgent to find the cause of bladder cancer recurrence and poor prognosis.Cancer stem cells(CSCs)have been shown to be an important factor in a variety of cancer progression and poor prognosis.Recent studies on its mechanism of action and biological functions have been increasingly explored and studied.In bladder tumors,research on cancer stem cells has been increasing in recent years,but complete screening from cancer stem cells to transcriptome analysis,screening of differential key factors are rare in bladder tumor research.Therefore,in this study,we isolated and identified bladder cancer stem cells(BCSCs),and then screened out key differentially expressed genes(DEGs)between tumor stem cells and their parental bladder cancer cells by transcriptome analysis.Based on this research,we screened out the key differential factors,and explored the biological mechanism of key factors in bladder tumors and its main affected protein signaling pathway to explore its detailed biological mechanism in bladder tumors.To provide a more complete theoretical basis for suggesting bladder tumor progression and poor prognosis.Methods:1.Two stable bladder cancer cell lines were selected as parental cell lines,and the bladder cancer stem cells were isolated and propagated by serum-free medium.In order to verify whether the cancer stem cells are cultured efficiently,the cancer stem cells are stably spheroidized,that is,when passaged to the third generation,total RNA is extracted,and examined the expression of several regulators of stemness and self?renewal activity by qRT?PCR.Animal experiments were performed to verify the difference in tumorigenic ability of tumor stem cells and their parental cell lines.2.Screening of differentially expressed genesTranscriptome analysis of cancer stem cells and their corresponding parental cell lines was performed by using Affymetrix HTA 2.0 Array.Differential gene analysis were performed by the GCBI Network Analysis Laboratory Platform(http://www.gcbi.com.cn).The differential genes of two different types of cancer stem cells and their parental cell lines were intersected,and the differential genes from different cell lines were screened as candidate differential genes.3.Correlation analysis between differentially expressed genes and prognosis of bladder cancer.The TCGA(GEPIA analysis platform)and ONCOMINE online public databases were used to query the expression of candidate differential genes in bladder tumors and normal bladder tissues,as well as the impact on bladder tumor survival rate.The factors with the most obvious difference in expression and having a great influence on the survival rate of bladder tumor were selected as the key factors.The pathologically diagnosed bladder tumor cases were randomly selected from local hospital,and the total RNA and total protein of cancer tissues and normal tissues were extracted.The expression of the selected factors was detected by PCR and Western blot.A variety of bladder tumor cell lines were cultured,and total RNA and total protein were extracted respectively.The expression of the selected factors was detected by PCR and Western blot.4.Experiments of key factors in bladder cancer cell linesCCK-8 proliferation assay,EdU assay,colony formation assay,cell cycle assay,cell sphere formation assay,apoptosis assay and real-time cell proliferation assay were performed to.clarify the role of the key factors in bladder cancer.5.The bladder tumor cell line was treated with a small molecule inhibitor,and the experimental group and the control group were subjected to transcriptomics and lipid metabolomics sequencing.Analysis of transcriptome differential pathways as well as differential fatty acids.Bladder cancer cells were injected subcutaneously in Balb/c nude mice(4-6weeks),and when a tumor of about 50 mm~3 was formed,small molecule inhibitors and differential fatty acids were used for gavage treatment for 3 weeks.The tumor size and weight of different groups were observed,and the transplanted tumors were taken for immunohistochemistry and Western blot to detect related proteinsResults:1.Two stable bladder cancer stem cells were successfully isolated and cultured by using serum-free medium.The mRNA expression levels of all five stemness factors are extremely up?regulated in BCSCs compared to their parental cells.Through in vivo experiments,it was found that the tumorigenic ability of cancer stem cells was significantly enhanced.2.By transcriptome analysis and differential expression gene analysis,a total of 17differentially expressed genes were obtained,namely ABCB1,SLC14A1,IL24,IFI44,DDX60,CXCL11,CLEC2B,SCD,RDH10,SLITRK6,PI3,IL7R,PAG1,DHRS3,C3,CDH2 and IL8.3.According to TCGA(GEPIA analysis platform),ONCOMINE online public database queries,among the 17 candidate differentially expressed genes,only SCD was significantly different in bladder tumor and normal bladder tissue,and had an adverse effect on the prognosis of patients with bladder cancer.4.By performing cell functional experiments,it was found that the treatment of bladder tumor cells with small RNA interference technology or small molecule inhibitors can significantly inhibit the proliferation ability of tumor cells,and to some extent inhibit the invasion and migration ability of bladder cancer cells,and bladder tumor does not show apoptosis due to the inhibition of SCD factor.5.Bladder cancer cell lines were treated with small-molecule inhibitors,and transcriptome and lipid metabolome sequencing tests were performed in the experimental group and the control group.Analysis found that SCD inhibitors mainly affect the Cell cycle,DNA replication,Cytokine-Cytokine receptor interaction,TNF signaling pathway,Fanconi anemia pathway,Homologous recombination,Bladder cancer and p53 signaling pathway.The nervonic acid was decreased obviously.Through the use of small molecule inhibitors of SCD and nervonic acid in animal models,it was found that compared with the blank control group,the tumor weight of the SCD small molecule inhibitor group showed a downward trend,and the tumor weight of nervonic acid intragastric administration group was significantly increased.Conclusion:SCD is an important marker of bladder cancer stem cells,and its high expression is correlated with the progression and poor prognosis of bladder cancer.Down-regulation of SCD activity can induce cell cycle arrest and effectively inhibit proliferation of bladder tumor cells by regulating cyclin-related proteins.In addition,partial inhibition of cell membrane function-related pathways also occurs.The inhibition of SCD activity reduced the synthesis of monounsaturated fatty acids in bladder cancer cells,and nervonic acid was significantly decreased. |
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