Investigating the tumor suppressive functions of the miR-34 family of microRNAs

描述(申请人提供)：microRNAs(MiRNAs)是作为转录后调节因子的非编码小RNA。MicroRNA失控是人类癌症的一个共同特征，大量的miRNA具有致癌或肿瘤抑制特性。MiR-34家族(miR-34a、miR-34b和miR-34c)受P53的直接调控，在体外可诱导细胞周期停滞和细胞凋亡，因而备受关注。重要的是，它的 在一些人类癌症中观察到了失活。尽管越来越多的证据表明miR-34具有肿瘤抑制作用，但以前对miR-34的研究大多是在体外进行的，或者使用非生理性表达水平的miR-34，这往往是人为的结果。因此，它在体内的确切功能、机制和功能相关靶点仍很大程度上是未知的。这些知识空白阻碍了对其进行治疗干预的利用。本应用的目的是研究miR-34家族的生理和肿瘤抑制特性。中心假设是miR-34家族的成员是p53反应的重要调节器，是真正的肿瘤抑制因子。该提案旨在回答以下问题：1)miR-34表达的慢性缺失是否促进体内肿瘤的发生？2)miR-34在P53通路中的生理作用是什么？3)哪些P53靶点与miR-34有很强的协同作用？为了回答这些问题，本应用程序建议利用我们实验室先前生成的miR-34家族的结构性和条件性基因敲除小鼠。在哺乳动物中，miR-34功能的完全遗传失活与生存能力是相容的，这为在生理背景下测试这些miRNAs的肿瘤抑制特性提供了独特的机会。初步结果表明，miR-34缺陷小鼠和细胞中依赖于p53的功能是完整的，这表明这个miRNA家族在p53途径中的功能可能是多余的或高度上下文特定的。因此，在本申请的第一个目的中提出的实验集中在两个人类癌症的小鼠模型上，其中已经描述了miR-34丢失，而第二个目标下的实验试图揭示miR-34和其他p53靶点之间的冗余。详细了解miR-34家族成员在正常和病理过程中的功能将是探索其肿瘤抑制特性和开发新的抗癌策略的关键一步。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are small, non-coding RNAs that act as post-transcriptional regulators. MicroRNA deregulation is a common feature of human cancers, and numerous miRNAs have oncogenic or tumor suppressive properties. The miR-34 family (miR-34a, miR-34b and miR-34c) has garnered much attention because it is directly regulated by p53 and can induce cell cycle arrest and apoptosis in vitro. Importantly, its inactivation has been observed in a number of human cancers. Despite the growing body of evidence suggesting its role as a tumor suppressor, most, if not all, previous studies on miR-34 have been done in vitro or using non-physiologic expression levels of miR-34, which are prone to artifactual results. Hence, its exact functions, mechanism and functionally relevant targets in vivo are still largely unknown. These gaps of knowledge prevent its exploitation for therapeutic intervention. The objective of this application is to investigate the physiologic and tumor suppressive properties of the miR-34 family. The central hypothesis is that members of the miR-34 family are important modulators of the p53 response, and are bona fide tumor suppressors. The proposal aims to answer the following questions: 1) Does chronic loss of miR-34 expression promote tumorigenesis in vivo? 2) What is the physiologic role of miR-34 in the p53 pathway? 3) Which p53 targets strongly cooperate with miR-34? To answer these questions, this application proposes to take advantage of constitutive and conditional knockout mice for the miR-34 family previously generated in our laboratory. Complete genetic inactivation of miR-34 function is compatible with viability in mammals, providing a unique opportunity to test the tumor suppressive properties of these miRNAs in a physiologic context. Initial results show that p53-dependent functions in miR-34-deficient mice and cells are intact, suggesting the functions of this miRNA family in the p53 pathway are likely redundant or highly context-specific. Thus, the experiments proposed in the first Aim of this application focus on two mouse models of human cancers wherein miR-34 loss has been described, while experiments under the second Aim seek to uncover redundancies between miR-34 and other p53 targets. The detailed understanding functions of members of the miR-34 family in normal and pathological processes that will emerge from the proposed study is a critical step towards not only exploring their tumor suppressive properties, but also developing novel anticancer strategies.