miR-29/miR-503/miR-489对长链非编码RNA-ncRAN靶向调控的鉴定及其提高膀胱癌细胞化疗敏感性作用的研究

MicroRNAs (MiRNAs) involve in the DNA damage signaling pathway activated by chemotherapeutic agents. However, the regulatory relations between these non-coding RNAs remain unknown. We previously identified and analyzed a long non-coding RNA, non-coding RNA expressed in aggressive neuroblastoma (ncRAN), that promoted the development of bladder tumor and was associated with resistance to chemotherapy. Further investigation showed that upon cisplatin (CDDP) treatment, 3 candidate miRNAs (miR-29, miR-503, and miR-489) targeting the 3'-untranslated region of ncRAN could inhibit ncRAN expression and increase the sensitivity of T24 cell to CDDP. On the basis of these data, we hypothesized that some specific miRNAs may increase the sensitivity of bladder cancer cells to CDDP by directly down-regulating expression of the long non-coding RNA, ncRAN. In this project, we aimed to a further investigate the direct targeted regulation of ncRAN by the candidate miRNAs through a series of molecular biology methods such as dual-luciferase reporter assay, immunofluorescence localization, real-time quantitative reverse transcription-polymerase chain reaction, mRNA half-life determination and gene microarray. Furthermore, in vitro and in vivo experiments were conducted to illustrate the molecular mechanism underlying the inhibition of ncRAN-conferred chemotherapy resistance by the candidate miRNAs. The findings of this study may have important implications in demonstrating the mechanism of post-transcriptional regulation of long non-coding RNAs and identification of new therapeutic targets for sensitizing chemotherapy for bladder cancer.

miRNA参与调节化疗药物激活的DNA损伤信号通路，但此过程中非编码RNA分子间的调控关系尚不清楚。申请者在前期工作中鉴定并解析了一个促进膀胱肿瘤恶性进展及化疗抗性的长链非编码RNA-ncRAN，进而发现顺铂处理后，三个与ncRAN3'UTR区序列完全匹配的miRNA分子（miR-29，miR-503 和miR-489）抑制ncRAN表达并促进T24细胞对顺铂的敏感性。由此我们提出假说：化疗诱导条件下，某些特异性的miRNA可通过直接下调ncRAN表达促进膀胱癌细胞对顺铂的敏感性。本研究拟进一步采用双荧光素酶报告基因分析、免疫荧光定位、mRNA定量表达及半衰期测定、基因芯片等分子生物学方法，结合体内、体外功能实验，旨在获得候选miRNA直接调控ncRAN的可靠证据。本项目将有助于阐明长链非编码RNA 的转录后调控机制，为鉴定膀胱癌化疗增敏的新靶点提供了更充分的科学依据。

miRNA 参与调节化疗药物激活的 DNA 损伤信号通路，但此过程中非编码 RNA 分子间的调控关系尚不清楚。申请者在前期工作中鉴定并解析了一个促进膀胱肿瘤恶性进展及化疗抗性的长链非编码 RNA-ncRAN，进而发现顺铂处理后，三个与 ncRAN3'UTR 区序列完全匹配的 miRNA 分子（miR-29，miR-503和 miR-489）抑制 ncRAN 表达并促进 T24 细胞对顺铂的敏感性。由此我们提出假说：化疗诱导条件下，某些特异性的 miRNA 可通过直接下调 ncRAN 表达促进膀胱癌细胞对顺铂的敏感性。本项目进一步采用双荧光素酶报告基因分析、免疫荧光定位、mRNA 定量表达及半衰期测定、基因芯片等分子生物学方法，结合体内、体外功能实验，发现如下重要结果：1.证明了 miR-29/miR-503/miR-489分子对ncRAN的直接靶向调控关系，并确定了具体的靶向结合位点；2.证明 了miR-29/miR-503/miR-489通过下调 ncRAN表达，抑制膀胱癌细胞存活、侵袭的作用；3.探索了miR-29/miR-503/miR-489 对 ncRAN 所介导的信号通路的影响，发现ncRAN可能作为ceRNA（竞争性内源RNA）通过miR-489调控了HDAC2、AKT3所涉及的促存活信号网络，进而促进了膀胱癌肿瘤细胞对CDDP的耐药性；4.利用活体动物模型和临床标本，初步阐明通过加入外源性 miR-29/miR-503/miR-489 下调ncRAN表达，促进膀胱癌细胞对顺铂敏感性的分子机制。确认ncRAN可作为提示膀胱癌进展及预后的独立预后因子。上述结果不仅从长链非编码 RNA转录后调控的崭新视角揭示了逆转膀胱癌耐药的分子机制，为针对非编码 RNA调控网络的分子靶向治疗提供重要靶点和有力科学依据，同时也为新兴的 ceRNA假说提供了有益补充