ABSTRACT
BACKGROUND: Bladder cancer stem cells could promote the recurrence and drug resistance of bladder cancer. Numerous studies have shown that keratin 6B (KRT6B) is involved in the production and progression of tumors, and is closely related to the prognosis of tumors. OBJECTIVE: To observe the expression of keratin 6B in CD44+ bladder cancer stem cells and to show the influence of keratin 6B on proliferation, migration, and self-renewal of bladder cancer stem cells, and to further explore the effect of keratin 6B expression on the prognosis of bladder cancer patients. METHODS: (1) CD44+ 5637 bladder cancer stem cells were isolated by magnetic active cell sorting. Cancer stem cell-related gene expression of SOX2, OCT4, and NANOG was detected via real-time polymerase chain reaction. The spheroid formation assay was used to detect the ability of self-renewal of cancer stem cells in CD44+ cells. Keratin 6B expression was detected in CD44+ bladder cancer stem cells by real-time polymerase chain reaction. (2) The CD44+5637 bladder cancer stem cells were divided into two groups. In the keratin 6B siRNA group, keratin 6B small interfering RNA was transfected into CD44+ bladder cancer stem cells. Untransfected CD44+ bladder cancer stem cells were used as the black control group. Cells were collected at 2 days post-transfection. The proliferation, migration, and self-renewal capacity of keratin 6B siRNA CD44+ bladder cancer stem cells were detected by the colony and wound healing assay and spheroid formation respectively. (3) Totally 24 bladder cancer tissues were used by immunohistochemistry to analyze the expression of CD44v6 and keratin 6B. (4) ONCOMINE database was used to analyze the effect of keratin 6B expression on the overall survival of bladder cancer. RESULTS AND CONCLUSION: (1) Cancer stem cell-related genes (SOX2, OCT4, NANOG) and keratin 6B expression was higher in CD44+ cells isolated by magnetic active cell sorting compared with CD44- cells (P < 0.05). Cell proliferation, migration, and in vitro spheroid formation were significantly increased (P < 0.05). Keratin 6B small interfering RNA down-regulated the expression of keratin 6B in CD44+ bladder cancer stem cells (P < 0.05). (2) Compared with the blank control group, the proliferation and migration of CD44+ bladder cancer stem cells after transfection of keratin 6B small interfering RNA (P < 0.05), and the number of tumorsphere significantly diminished (P < 0.05); the expression of Notch1 and Hes1 mRNA increased (P < 0.05). (3) Keratin 6B and CD44v6 were significantly different in bladder cancer tissue (P=0.006). The overall survival rate of bladder cancer patients with high expression of keratin 6B was lower than that of patients with low expression of keratin 6B. (4) The results showed that keratin 6B was highly expressed in CD44+ bladder cancer stem cells, and could promote the proliferation, migration, and self-renewal capacity of bladder cancer stem cells. The high expression of keratin 6B contributes to improving the survival of bladder cancer patients.
ABSTRACT
Objective To investigate the expression profile and biological function of lncRNAs in urothelial cancer stem cells and explore whether they can be biomarkers for prediction of clinical characteristics for bladder cancer patients.Methods Microarray analysis was performed to identify differentially expressed lncRNAs in cancer stem cells and parental cancer cells.Expression profiles were validated by Coding Potential Caculator analysis,real-time polymerase chain reaction.By performing in vitro sphere formation assays,J82 cells with lentivirus-based knockdown of lncRNA-BCSC (bladder cancer stem cells) were used to confirmed its function.Samples were obtained from patients who underwent TURBT between January 2009 and December 2012.All tissues were initially confirmed by pathologists.The association of the clinicpathologic of bladder cancer and lncRNA-BCSC expression was analyzed by x2 analysis.Results 750 lncRNAs were highly expressed from microarray analysis in bladder cancer stem cells.Among these,lncRNA-BCSC was identified as potentially maintaining self-renewal of cancer stem cells.Knockdown of this transcript in J82 cells inhibited spheroid formation.lncRNA-BCSC expression were higher in 54.8％ (57/104) bladder cancer tissues.Moreover,using x2 analysis,lncRNA-BCSC expression in primary tumors was found to be a predictor for recurrence following transurethral resection in patients with nonmuscle-invasive bladder cancer (P =0.009).Conclusions Our study provides strong evidence that lncRNA-BCSC are indispensable modulators of self-renewal ability of bladder cancer stem cells.Overexpression of lncRNA-BCSC may have a predictive value for early recurrence in patients suffering from nonmuscle-invasive bladder cancer.