miR-191在苯并芘诱导DNA损伤应激反应中的功能研究

Cellular DNA damage response (DDR) is a very complex process, which requires the involvement of many signaling pathways and proteins/molecules. However, the underlying molecular mechanism is still far from clear. Previously, we have found the expression of KHSRP protein, which participates in the maturation of microRNA(miRNA), was changed after genotoxic agent treatment in a proteomic study. miRNA array showed that the expression levels of many miRNAs were indeed changed by BaP treatment, suggesting that miRNAs possibly was involved in the DDR. In this study, we focus on miR-191, which was identified in the miRNA array experiment, in an effort to understand its function in DDR. First, we will examine how BaP treatment leads to the expression change of miR-191, and then the involvement of miR-191 in DDR will be explored. Finally, which pathway miR-191 employed to participate in the DDR will be identified. The study will help us elucidate the role of miR-191 in the DDR process induced by BaP. It will also provide a better understanding of the DDR process induced by environmental pollutant, and some scientific evidence for the carcinogenesis after exposure of BaP.

细胞的DNA损伤应激反应是一个很复杂的过程，研究已发现有多条信号通路和多种蛋白/分子参与这一过程，但其分子机制还远未明确。我们在前期应用蛋白组学研究DNA损伤剂诱导的DDR过程中，发现一个参与microRNA（miRNA）成熟的蛋白KHSRP的表达发生改变。进一步miRNA芯片结果证明DNA 损伤剂BaP处理可诱导多种miRNA的表达改变，提示miRNA极有可能参与了其中的DDR。本研究在前期工作基础上，针对芯片结果中表达发生改变的miR-191展开进一步的工作，首先明确BaP如何诱导miR-191表达改变；其次分析miR-191是否参与了DDR过程；最后尝试阐明miR-191是通过何种通路参与了细胞的DDR过程。通过以上研究，将明确miR-191在BaP诱导的DDR过程中发挥的作用。使我们对环境污染物诱导的DDR过程有更深一步的认识，也为探索BaP致癌机制提供一定的科学证据。

前期研究首先采用了microRNA芯片分析了细胞对苯并芘的应答反应，并发现了一批表达改变的microRNA。从中我们选择了miR-191进行了进一步的功能研究。结果发现过表达miR-191抑制了苯并芘处理的细胞增殖，加重苯并芘诱导的DNA损伤，并引起了细胞阻滞于G2/M期，而苯并芘诱导的凋亡未受影响。通过数据库预测和双荧光素酶报告系统，我们鉴定了RCC2和CDK6为miR-191的靶标。相应地，干扰RCC2后，苯并芘处理的细胞表现出和过表达miR-191相同的趋势。进一步我们又构建了RCC2过表达质粒，并进行免疫沉淀和质谱鉴定，发现了一批和RCC2有潜在互作的蛋白。其与Ku80的相互作用通过免疫共沉淀得到证实。综上所述，苯并芘诱导DNA损伤后，miR-191表达上调，进而下调其靶标RCC2，从而抑制了苯并芘处理的细胞增殖，加重苯并芘诱导的DNA损伤，并引起了细胞阻滞于G2/M期。其作用可能和DNA损伤修复中的重要蛋白Ku80相关。我们初步得出了RCC2在苯并芘诱导的DNA损伤中发挥重要的作用，为进一步理解DNA损伤通路提供了更丰富的信息。