Exploring miR-29 in melanoma progression and prevention

项目摘要 miR-29家族的microRNA（miRNAs），由miR-29 a、miR-29 b1、miR-29 b2和miR-29 c组成， 由两个miRNA簇（miR-29 a ~b1和miR-29 b2 ~c）编码，在肿瘤细胞中具有肿瘤抑制特性。 各种人类癌症。我们发现，自相矛盾的是，致癌BRAFV 600 E诱导转录， 通过MAPK通路激活（miR-29 a ~b1）或致癌应激介导的黑素细胞簇 p53激活（miR-29 b2 ~c）。这就形成了一个阻止黑色素瘤发展的屏障， 用海绵构建体灭活miR-29家族促进的基因工程小鼠模型 黑素瘤进展。有趣的是，来自痣的工程人类细胞系模型和人类表达数据 而黑色素瘤显示黑色素瘤的进展与miR-29 b2 ~c的表达降低有关。 基于这些发现，我们假设关键miR-29靶点的去抑制和过表达 促进黑色素瘤的发展，这些目标是黑色素瘤的弱点，可以利用 治疗我们建立了一个高通量的基因工程小鼠模型平台， miR-29靶点在黑色素瘤中的作用，并评估其作为治疗靶点的潜力。在本提案中，我们 将广泛使用这个平台，首先测试是否恢复miR-29表达，从而抑制所有miR-29的表达。 29个靶点损害黑色素瘤的发展。我们将进一步评估单个miR-29靶点的沉默是否会导致 对黑色素瘤的发生有显著影响。我们已经鉴定出致癌转录因子MYBL 2是一种肿瘤抑制因子， 真正的miR-29靶点，其沉默降低了体外黑色素瘤细胞系的侵袭性。我们将测试是否- 在黑色素瘤小鼠模型中，抑制的MYBL 2通过沉默MYBL 2介导miR-29丢失的作用 miR-29失活。此外，我们将在具有正常miR-29活性的模型中沉默MYBL 2，以确定 MYBL 2是黑色素瘤的一个普遍弱点。为了确保与人类黑素瘤的相关性，我们将补充 这些基因工程小鼠模型与人类黑色素瘤PDX的实验。我们建议的研究 将使用我们新开发的小鼠建模平台作为管道，系统地评估miR-29是否靶向 一般而言，特别是MYBL 2，具有作为黑色素瘤治疗靶点进一步探索的潜力。

PROJECT SUMMARY The miR-29 family of microRNAs (miRNAs), consisting of miR-29a, miR-29b1, miR-29b2, and miR-29c, is encoded by two miRNA clusters (miR-29a~b1 and miR-29b2~c) and has tumor suppressive properties in a variety of human cancers. We found that, paradoxically, oncogenic BRAFV600E induces the transcription of both clusters in melanocytes through either MAPK pathway activation (miR-29a~b1) or oncogenic stress-mediated p53 activation (miR-29b2~c). This creates a barrier that prevents melanoma development, and we showed in a genetically engineered mouse model that inactivation of the miR-29 family with a sponge construct promotes melanoma progression. Interestingly, engineered human cell line models and human expression data from nevi and melanomas show that melanoma progression is associated with decreased expression of miR-29b2~c. Based on these findings we hypothesize that de-repression and overexpression of critical miR-29 targets promotes melanoma development and that such targets are vulnerabilities of melanoma that can be exploited for therapy. We have established a high-throughput genetically engineered mouse modeling platform to evaluate the role of miR-29 targets in melanoma and to assess their potential as therapeutic targets. In this proposal, we will extensively use this platform to first test if restoration of miR-29 expression and, thus, repression of all miR- 29 targets impairs melanoma development. We will further evaluate if silencing of a single miR-29 target elicits significant effects on melanomagenesis. We have identified the oncogenic transcription factor MYBL2 as a bona fide miR-29 target whose silencing reduces the aggressiveness of melanoma cell lines in vitro. We will test if de- repressed MYBL2 mediates the effects of miR-29 loss by silencing MYBL2 in a melanoma mouse model driven by miR-29 inactivation. Moreover, we will silence MYBL2 in a model with normal miR-29 activity to determine if MYBL2 is a general vulnerability of melanoma. To ensure relevance to human melanoma, we will complement these genetically engineered mouse models with experiments in human melanoma PDXs. Our proposed studies will use our newly developed mouse modeling platform as a pipeline to systematically evaluate if miR-29 targets in general, and MYBL2 specifically, have the potential to be further explored as therapeutic targets in melanoma.