miR-21 signaling in tumor response to radiation treatment

描述（由申请人提供）：克服辐射抗性对于改善放射治疗至关重要。近年来，我们发现miR-21作为一种癌基因miR，通过促进DNA双链修复参与细胞的辐射抗性。这些结果表明，靶向miR-21是使肿瘤细胞对辐射增敏的良好策略;然而，由于辐射可以诱导miR-21上调，因此上调的miR- 21反过来可以降低单独靶向miR-21对辐射增敏的作用。此外，周围肿瘤组织中的miR-21状态可能影响肿瘤对电离辐射治疗的反应，因为肿瘤微环境是影响肿瘤情况的重要因素。因此，为了有效地阻断miR-21在肿瘤放射抵抗中的作用，我们需要了解miR-21在体外和体内辐射肿瘤细胞中信号转导的全貌。为此，我们设计了两个目标， 这个提议。目的1：确定IR如何刺激miR-21上调。我们将用分子和细胞生物学的方法和手段来阐明IR促进的miR-21上游调控，主要回答以下四个问题：1）IR激活的ATR是否通过磷酸化STAT 3在S694位点来刺激miR-21的表达。2)STAT 3作为转录因子是否能直接刺激EGFR的表达以响应IR; 3）EGFR是否通过磷酸化AGO 2 Y393来抑制miR-21的成熟以响应IR; 4）miR-21是否能直接靶向p53，p53通过抑制EGFR的转录因子YY 1和SP1来负调控EGFR的表达。目的2：确定肿瘤微环境中的miR-21是否以及如何影响肿瘤对IR治疗的反应。我们将使用两种小鼠模型：1）miR-21敲入或敲除小鼠模型：我们将使用可以在这些免疫有效小鼠中生长肿瘤的肿瘤细胞。2)裸鼠模型：我们将使用能够在免疫缺陷小鼠中生长异种移植肿瘤的人肿瘤细胞。这些肿瘤细胞系（小鼠或人）将用miR-21或其上游调节剂下调，皮下接种到小鼠的后腿中。我们将在肿瘤区域照射后观察肿瘤大小，并检查肿瘤组织和周围组织中miR- 21/其相关调控因子的水平/活性。该研究结果有望帮助我们更好地了解miR-21在体内外介导肿瘤放射抵抗中的作用，从而为在不久的将来阻断miR-21通路以改善放射治疗提供一种有效的方法。

DESCRIPTION (provided by applicant): Overcoming radioresistance is essential for improving radiotherapy. Recently, we found that miR-21 as an onco-miR involved cell radioresistance through promoting DNA double strand repair. These results suggest that targeting miR-21 is a good strategy for sensitizing tumor cells to radiation; however, since radiation could induce miR-21 up-regulation, the up-regulated miR- 21 could in turn reduce the effects of targeting miR-21 alone on radiosensitization. In addition, the miR-21 status in the surrounding tumor tissue may affect tumor response to ionizing radiation treatment since the tumor microenvironment is an important factor that affects the tumor situation. Therefore, in order to efficiently block the function of miR-21 in tumor radioresistance, we need to know the whole picture of miR-21 signaling in irradiated tumor cells in vitro and in vivo. For this purpose, we designed two aims in this proposal. Aim 1: Determine how IR stimulates miR-21 up-regulation. We will elucidate IR-promoted upstream regulation of miR-21 with the methods and approaches of molecular and cellular biology by answering the following four questions: 1) Whether the IR-activated ATR stimulates miR-21 expression through phosphorylating STAT3 at S694. 2) Whether STAT3 as a transcription factor could directly stimulate the EGFR expression to respond to IR. 3) Whether EGFR inhibits miR-21 maturation through phosphorylating AGO2 at Y393 to respond to IR. 4) Whether miR-21 could directly target p53 that negatively regulates EGFR expression through inhibiting YY1 and SP1 (the transcription factors of EGFR). Aim 2: Determine whether and how miR-21 in the tumor microenvironment affects the tumor response to IR treatment. We will use two mouse models: 1) miR-21 knock-in or knockout mouse model: we will use tumor cells that can grow tumors in these immune efficient mice. 2) nude mouse model: we will use the human tumor cells that can grow xenograft tumors in the immune deficient mice. These tumor cell lines (mouse or human) will be down-regulated with miR-21 or its upstream regulators, subcutaneously inoculated into the hind legs of the mice. We will observe the tumor size and examine the level/activity of miR- 21/its relevant regulators in the tumor tissue and the surrounding tissues after the tumor areas are irradiated. The results from this proposal are expected to help us better understand the role of miR-21 in mediating tumor radioresistance in vitro and in vivo, thus, providing an efficient way to block the miR-21 pathway for improving radiotherapy in the near future.