miR-449a 通过靶向下调c-Myc 增强肿瘤细胞辐射敏感性的机制研究

c-Myc was one of the first proto-oncogenes identified and its overexpression at the RNA and protein levels has subsequently associated with a wide variety of human cancers. It has been known that c-Myc amplification not only contributes to the genesis and progression of most human cancers, but affects the outcome of cancer radio- or chemotherapy. Radiation resistance of cancer cells was strongly linked to the overexpression of c-Myc. But the molecular mechanism underlying c-Myc-induced cancers not completely understanding. miR-449a is evolutionarily conserved, endogenous, small noncoding RNA that regulate oncogenes encoding protein. miR-449a is involved in the tumour development, progression and response to therapy. It has been found miR-449a was downregulated in various cancers,including gastric, lung, liver and gynecologicla cancers. Thus, in this study, on the one hand, we will ascribe the overexpression of c-Myc to miR-449a downregulation or deletion in human cancer cells and provide evidence c-Myc is a direct target of miR-449a. on the other hand, we will further show that miR-449a enhanced the radiosensitivity of prostate cancer cells by targeting c-Myc. we hope we can demonstrate that mechanism of miR-449a-targeted c-Myc to controll the radiosensitivity of cancer cells. This study will reveal a previously unrecognized function of miR-449a-mediated c-Myc regulation in response to IR, and will provide alternative therapeutic strategies in the radiation treatment of human cancer.

c-Myc是第一个被鉴定的原癌基因。目前已知，其RNA和蛋白水平的过表达与人类肿瘤相关。c-Myc不仅参与调控人类肿瘤的起始和进展，而且影响多个恶性肿瘤的放/化疗效果。但其诱导肿瘤发生及放/化疗耐受的分子机制并不清楚。miR-449a是一个新发现的内源性、小非编码RNA分子。已有研究表明，miR-449a可以通过靶向调控多个原癌基因，参与各类肿瘤的起始、侵袭和转移。已知，miR-449a在人类多种肿瘤中表达下调或缺失，包括胃癌、肺癌、肝癌及妇科癌症等。因此，本项目一方面将人类癌细胞中c-Myc的过表达与miR-449a的下调或缺失联系起来，提出c-Myc是miR-449a调控的一个直接靶基因，阐明 miR-449a的下调或缺失是c-Myc过表达的一个关键因素。另一方面，深入研究miR-449a通过下调c-Myc增强肿瘤细胞辐射敏感性的分子机制。希望通过对其分子机制的阐明，找到肿瘤细胞辐射增敏的新靶点，为小分子增敏药物研发提供新思路。

作为转录后基因表达调控因子，miRNAs影响多个信号传导通路，包括电离辐射应答。为揭示miRNA在肿瘤细胞电离辐射应答中的表达变化及作用，阐明miRNA影响肿瘤细胞辐射敏感性的分子机制，本项目首次采用人源miRNA microarray技术筛选出电离辐射后前列腺癌细胞中差异表达miRNAs。随后进一步采用荧光定量PCR进行验证。结果显示，电离辐照后，miR-449a表达显著上升。采用TargetScan预测c-Myc是miR-449a的一个靶基因。双荧光素酶报告系统证实在LNCaP细胞中miR-449a通过与c-Myc 3‘-UTR结合靶向抑制c-Myc表达。在miR-449a过表达LNCaP细胞，IR诱导的细胞生长阻滞增强，敏感性提高。RNAi技术敲低c-Myc得到相同结果。这说明，miR-449a在LNCaP细胞中可通过靶向下调c-Myc增强癌细胞的辐射敏感性。. 但进一步的研究发现，miR-449a/c-Myc在肿瘤细胞电离辐射应答中的作用并不广泛。在Rb野生型的LNCaP和PC-3细胞中过表达miR-449a，辐射敏感增加；但在Rb突变的DU-145细胞中过表达miR-449a，辐射敏感性并不增加。推测miR-449a增强前列腺癌细胞的辐射敏感性可能与RB蛋白状态相关。进一步采用WB分析细胞周期和凋亡相关的信号蛋白表达发现，miR-449a/c-Myc增强前列腺癌细胞的辐射敏感性与CDC25A/RB/E2F1信号通路相关。这些研究结果为探讨前列腺癌的发生发展分子机理提供了新思路，考虑到miR-449a增强前列腺癌细胞辐射敏感性的潜能，本文的研究结果可为前列腺癌的放射治疗提供一个新靶点。