长链非编码RNA LOC572558对膀胱癌生物学行为的调控及其机制研究

The long non-coding RNA (lncRNA) can regulate the biological behavior of tumor cells through the regulation of gene expression. It is also closely correlated with tumor recurrence, metastasis and prognosis. However, the lncRNA expression spectrum in bladder cancer and its mechanism of action remain unknown. We used lncRNA chips to screen aberrantly expressed lncRNAs in bladder cancer and identified a highly up-regulated lncRNA LOC572558 by functional experiments. By EMSA experiment, we also found that LOC572558 may be combined with the EZH2 protein. Therefore, we believe that LOC572558 possibly act through the combination with EZH2 protein to recruit PRC2 protein complex, thereby regulating the expression of downstream target genes. We will clarify whether LOC572558 is combined with EZH2 protein by RNA co-immunoprecipitation and RNA pull-down. By deletion mapping experiment, we can find its specific binding site. Next, we will establish bladder cancer cell lines with different expression levels of LOC572558 and EZH2, and apply western blot and CHIP to demonstrate whether the PRC2 complex is the downstream target gene of LOC572558. We aim to clarify the mechanism by which LOC572558 regulate bladder cancer biological behavior, and hoping to find a promising target for the treatment of bladder cancer.

长链非编码RNA（lncRNA）可以通过调控基因表达影响肿瘤细胞的生物学行为，并与肿瘤的复发、转移和预后密切相关。然而目前膀胱癌中lncRNA表达谱及其作用机制的研究尚处于空白状态。我们通过lncRNA芯片和功能预实验筛选出在膀胱癌中高表达的 LOC572558，通过EMSA实验发现其可能与EZH2蛋白结合。因此我们认为LOC572558可能通过结合EZH2蛋白来招募PRC2复合体，进而调节下游靶基因的表达。我们将采用RNA免疫共沉淀和RNA pull-down技术明确LOC572558是否同EZH2相结合，然后通过缺失作图技术找到其结合位点。接下来建立LOC572558和EZH2不同表达水平的膀胱癌细胞系，采用Western blot和CHIP等手段论证PRC2是否为LOC572558的下游靶基因。最终明确LOC572558在膀胱癌中的生物学功能及其作用机制，希望为膀胱治疗提供新的靶点

长脸非编码RNA在包括膀胱癌的多种肿瘤的发生和进展中起到重要作用。在本研究中，我们选择了在膀胱癌中异常表达的lncRNA LOC572558进行功能学的探索。我们首先通过慢病毒感染构建了过表达LOC572558的膀胱癌细胞稳转株，进一步通过CCK-8实验、流式细胞仪、细胞划痕实验及Transwell实验等手段检测了过表达LOC572558对膀胱癌细胞生物学行为的影响，结果发现过表达LOC572558可以抑制膀胱癌T24和5637细胞的增殖，诱导膀胱癌细胞发生凋亡，使膀胱癌细胞阻滞于S期，降低G1期细胞的比例，并且抑制膀胱癌细胞的侵袭和迁移能力。进一步通过蛋白芯片和westernblot试验我们发现，LOC572558可以调节AKT、MDM2和P53等信号通路蛋白的磷酸化水平。综合上述结果，我们认为LOC572558在膀胱癌中通过调节P53信号通路发挥了抑癌基因的作用，可能成为未来膀胱癌一个诊断和治疗的新靶点。