干性相关长链非编码RNA促进膀胱癌干细胞自我更新及其作为经尿道膀胱肿瘤切除术后复发预测指标的研究

Stem cell associated IncRNA promotes the self-renewal in bladder cancer stem cells and acts as a predictor for recurrence of bladder cancer

目的 鉴别和筛选出促进膀胱癌干细胞（bladder cancer stem cells,BCSC）自我更新的长链非编码RNA（long non-coding RNAs,lncRNAs）,并进一步分析其预测膀胱癌复发的价值.方法 2012年3月至2015年1月通过悬浮球囊培养法获得膀胱癌J82球囊及食管癌KYSE150球囊,以相应的肿瘤细胞作为对照,采用基因芯片技术及在线软件（coding potential caculator,CPC）分析检测两种球囊及细胞中具有差异表达的lncRNAs,筛选出表达量最高的lncRNAs.荧光定量PCR法验证lncRNAs在球囊中的表达量.采用慢病毒shRNA技术构建稳定下调lncRNA-BCSC表达的膀胱癌J82细胞株,观察其对膀胱癌球囊形成能力的影响.选取2009年1月至2012年12月收治的104例膀胱癌患者,行经尿道膀胱肿瘤切除术（TURBT）并经病理检查确诊为膀胱癌.采用荧光定量PCR法检测膀胱癌组织和正常膀胱黏膜组织中lncRNA-BCSC的相对表达量,分析lncRNA-BCSC相对表达量与临床病理指标的相关性.结果 经悬浮球囊培养法获得膀胱癌J82球囊,经基因芯片技术鉴定出750个高表达的lncRNAs,CPC非编码转录本评分及荧光定量PCR法验证筛选出lncRNA-BCSC.稳定下调lncRNA-BCSC的J82细胞的球囊形成能力受到抑制.104例膀胱癌组织中,57例（54.8％）IncRNA-BCSC高表达.高表达的lncRNA-BCSC与膀胱癌患者TURBT术后复发率呈正相关（P=0.009）.结论 lncRNA-BCSC能够促进BCSC自我更新,lncRNA-BCSC高表达与TURBT术后膀胱癌复发率呈正相关,有望成为预测膀胱癌复发与精准治疗的分子标志物.

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.