ATR为核心的microRNA调控网络及miR-185对肿瘤细胞辐射增敏的研究

ATR is an important protein kinase in cells responding to radiation stress. However,it is still unclear which microRNA involves in the regulation of ATR. In our previous work, it was found that miR-185 targeted ATR and increased the radiosensitivity of tumor cells. Based on these findings, point mutation, cell line with inducible miR-185 expression and inhibitors will be used to verify the targeted regulation of miR-185 on ATR and its downstream gene expressions. The role of ATR　in the generation of microRNAs will be investigated. The regulation network based on ATR responding to radiation will be optimized. Finally, the possibility of increasing radiosensitivity of tumor cells through down-regulation of ATR by miR-185 will be tested by cell and animal experiments. The obtained results not only elucidate the importance of biological molecular interaction in cellular signalling transduction, but also provide new possible option for cancer therapy.

ATR是细胞中参与辐射应激响应的重要激酶，但目前对调控ATR的microRNA仍一无所知。我们的前期工作提示，miR-185对ATR具有靶向调控作用并能增加肿瘤细胞的辐射敏感性。本项目拟在此基础上采用点突变技术、诱导表达miR-185的细胞系、抑制剂等方法明确miR-185对ATR及其下游基因表达的靶向调控作用，并探讨ATR在microRNA发生中的作用，完善以ATR为核心的辐射生物效应的调控通路。最后通过细胞实验和动物实验探讨利用miR-185对ATR的负调控进行肿瘤细胞辐射增敏的可能性。研究结果不仅有助于阐明生物分子间的相互作用在细胞信号传导中的重要性，还能为肿瘤治疗提供新的选择途径。

本研究通过荧光报告载体实验、点突变技术、实时荧光定量PCR、western blotting等方法首次发现了miR-185对ATR的靶向调控作用，明确了miR-185通过对ATR及其下游基因表达的调控参与辐射应激响应过程。通过转染ATR特异siRNA、深度测序等方法对ATR-miRNA调控网络进行研究，得到了较完整的以ATR为核心的miRNAs及辐射损伤修复因子相互作用的网络系统，包括miR-3928-Dicer-miR-185、miR-663、miR-300、miR-4767、miR-378d 等miRNA调控网络，及以CHK1、P53、BRCA1、RAD17 等与DNA损伤感受、修复密切相关的蛋白。细胞实验和动物实验表明：miR-185通过提高辐射诱导的凋亡细胞比率和抑制细胞增殖速率来增加肿瘤细胞的辐射敏感性。这些研究结果不仅有助于揭示miRNA表达调控的新机制，并且为利用以ATR为核心的调控网络进行肿瘤辐射增敏提供了大量的实验和理论依据，有望为肿瘤治疗方案提供新的靶点，在肿瘤放疗中具有广阔的应用前景。