Background: Stem cells characterized by self-renewal and therapeutic resistance play crucial roles in bladder cancer (BLCA). However, the genes modulating the maintenance and proliferation of BLCA stem cells are still unclear. In this study, we aimed to characterize the expression of stem cell-related genes in BLCA. Methods: The mRNA expression-based stemness index (mRNAsi) of The Cancer Genome Atlas (TCGA) was evaluated and corrected by tumor purity. Corrected mRNAsi were further analyzed with regard to muscle-invasive bladder cancer molecular subtypes, survival analysis, pathological staging characteristics, and outcomes after primary treatment. Next, weighted gene co-expression network analysis was used to find modules of interest and key genes. Functional enrichment analysis was performed to functionally annotate the modules and key genes. The expression levels of key genes in all cancers were validated using Oncomine and Gene Expression Omnibus (GEO) database containing molecular subtypes in BLCA. Protein interaction networks were used to identify upstream genes, and the relationships between genes were analyzed at the protein and transcription levels. Findings: mRNAsi was significantly upregulated in cancer tissues. Corrected mRNAsi in BLCA increased as tumor stage increased, with T3 having the highest stem cell characteristics. Lower corrected mRNAsi scores had better overall survival and treatment outcome. The modules of interest and key genes were determined based on topological overlap measurement clustering results and the inclusion criteria. For 13 key genes ( AURKA, BUB1B, CDCA5, CDCA8, KIF11, KIF18B, KIF2C, KIFC1, KPNA2, NCAPG, NEK2, NUSAP1 , and RACGAP1 ), enriched gene ontology terms related to cell proliferation (e.g., mitotic nuclear division, spindle, and microtubule binding) were determined. Their expression did not differ according to the pathological stages of BLCA, and these genes were clearly overexpressed in many types of cancers. In GEO database, the expression levels of 13 key genes were higher in basal subtype with the highest stem cell characteristics than in luminal and its subtypes. AURKB and PLK1 may be regulated upstream of other key genes, and the key genes were found to be strongly correlated with each other and with upstream genes. Interpretation: The 13 key genes identified in this study were found to play important roles in the maintenance of BLCA stem cells. Controlling the upstream genes AURKB and PLK1 may have applications in the treatment of BLCA. These genes may act as therapeutic targets for inhibiting the stemness characteristics of BLCA.

Background:The study was aimed to investigate the influence of circ_0058063 on tumorigenesis as well as the regulatory mechanism of circ_0058063/miR-145-5p/ CDK6 pathway in bladder cancer (BC).Methods: Bioinformatics analysis was used to screen highly expressed circle RNA (circRNA) and search its downstream microRNA (miRNA) and protein. The expression level of circRNA, miRNA, and CDK6 in BC cell lines T24 and J82 were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Small interfering RNA was used to downregulate circ_0058063 expression. Cell proliferation, cell cycle, cell apoptosis, and cell migration of T24 cells and J82 cells were detected through MTT assay, flow cytometry, and wound-healing assay, respectively.The relationships among miR-145-5p, circ_0058063, and CDK6 were confirmed through dual luciferase reporter assay. In vivo experiment was also performed to explore the impact of circ_0058063/miR-145-5p/CDK6 pathway on tumorigenesis in BALB/c nude mice.Results: Circ_0058063 was significantly overexpressed in BC tissues. The downregulation of circ_0058063 impaired BC cell proliferation and migration ability but improved cell apoptosis ability. Circ_0058063 repressed miR-145-5p, which inhibited the expression of CDK6. Downregulation of circ_0058063 or miR-145-5p transfection contributed to more cells arresting in G0/G1 stage. MiR-145-5p suppressed cell growth and migration ability in BC, whereas CDK6 exerted the opposite influence on these cellular events. In vivo experiment also indicated that tumor development in BALB/c nude mice was repressed remarkably when circ_0058063 was downregulated.Conclusion: Circ_0058063 acted as a sponge of miR-145-5p to promote BC progression by regulating CDK6 expression, which provided some potential targets for BC treatment. K E Y W O R D S bladder cancer, CDK6, circ_0058063, miR-145-5p, sponge

To investigate the anti-proliferative and chemosensitizing effects of luteolin on human gastric cancer, gastric cancer AGS cells were treated with luteolin and/or other chemotherapeutic agents. Cell growth was assessed by MTT assay, cell cycle and apoptosis were assessed by flow-cytometric analysis, and the expression of major proteins regulating cell cycle and apoptosis was also detected. The results showed that luteolin inhibited the growth of gastric cancer cells in a dose- and time-dependent manner. Flow cytometry revealed that the percentage of cells at G2/M phase increased dose-dependently. The protein levels of Cdc2, Cyclin B1 and Cdc25C were reduced and p21/cip1 was up-regulated after the treatment with luteolin. Furthermore, luteolin induced apoptosis in gastric cancer AGS cells. Western blotting showed that luteolin treatment significantly increased the levels of pro-apoptotic proteins, including Caspase-3, 6, 9, Bax, and p53, and decreased the levels of anti-apoptotic protein Bcl-2, thus shifting the Bax/Bcl ratio in favor of apoptosis. It was also demonstrated that a combinational treatment of cisplatin and luteolin induced more effectively cell growth inhibition, compared to cisplatin treatment alone. These findings indicate the anti-proliferative and chemosensitizing effects of luteolin on human gastric cancer AGS cells and luteolin may be a promising candidate agent used in the treatment of gastric cancer.

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