Non-coding RNA functions in tumor metastasis

尽管在对抗癌症方面已经取得了很大的进展，特别是在其早期阶段，但转移 仍然是一项艰巨的、往往是致命的挑战。很明显，非编码RNA，包括 microRNA和长链非编码RNA（lncRNA）是调节转移的分子网络的组分。 已知一些lncRNA具有与其基因组位点相反的功能;例如，相反的表型 已经报道了lncRNA Haunt基因缺失和插入失活，有趣的是， Haunt基因缺失效应是由于基因组DNA的丢失而不是Haunt RNA的丢失。因此，一个主要的 lncRNA研究的挑战是，缺失或失活lncRNA基因所导致的表型是否可以 可以明确地归因于lncRNA本身的丢失或重叠调控的丢失， 元素MALAT 1（转移相关肺腺癌转录物1）是最丰富的， 在正常组织中是保守的lncRNA，并且先前已被描述为转移启动子。然而，在这方面， 没有证据表明先前报道的Malat 1基因缺失（导致多个 Malat 1的邻近基因）或反义RNA（其从未通过拯救实验或通过 MALAT 1基因敲除细胞）的影响是特异性的Malat 1 lncRNA损失。出乎意料的是，使用转录 终止子插入策略，我们发现破坏Malat 1基因而不改变其表达， 乳腺癌转基因小鼠模型中的相邻基因显著促进了肺转移， 重要的是，这种表型被Malat 1的遗传加回完全逆转。CRISPR-Cas9- 在人乳腺癌细胞中介导的MALAT 1基因敲除诱导了它们的转移能力， Malat 1重新表达。相反，Malat 1的过表达抑制了乳腺癌的转移， 转基因小鼠和异种移植模型。从机制上讲，我们使用了最近开发的染色质分离， RNA纯化-质谱（ChIRP-MS）方法鉴定TEAD家族成员作为结合蛋白 在原发性乳腺肿瘤组织的内源性水平上对MALAT 1进行了研究，发现MALAT 1结合， 螯合并灭活促转移转录因子TEAD。我们还发现了 MALAT 1水平与乳腺癌进展和转移的关系。基于这些重要发现，我们 建议全面表征乳腺中MALAT 1的功能丧失和功能获得效应 癌症转移，使用基因工程小鼠模型，移植模型，同系模型， 异种移植模型和CRISPR-Cas9基因组编辑方法（具体目标1）;我们还将阐明 MALAT 1调节转移的机制（特异性目的2）。这一项目将导致对 目前高度丰富和保守的lncRNA模型，并将深刻推进理解 lncRNA在肿瘤转移中的功能和作用机制。

Although great advances have been made in combatting cancer, particularly at its early stages, metastasis remains a formidable and frequently fatal challenge. It has become evident that non-coding RNAs, including microRNAs and long non-coding RNAs (lncRNAs), are components of molecular networks regulating metastasis. Some lncRNAs are known to have opposing functions to their genomic locus; for instance, opposite phenotypes have been reported from the lncRNA Haunt gene deletion and insertional inactivation, and interestingly, the Haunt gene deletion effect was due to the loss of the genomic DNA but not the loss of Haunt RNA. Thus, a major challenge in lncRNA research is whether phenotypes resulting from deleting or inactivating a lncRNA gene can be unequivocally attributed either to the loss of the lncRNA per se or to the loss of overlapping regulatory elements. MALAT1 (metastasis associated lung adenocarcinoma transcript 1) is among the most abundant and conserved lncRNAs in normal tissues, and has previously been described as a metastasis promoter. However, there is no evidence that the previously reported Malat1 gene deletion (which led to upregulation of multiple Malat1's adjacent genes) or antisense RNA (which has never been validated by rescue experiments or by MALAT1 knockout cells) effect was specific to Malat1 lncRNA loss. Unexpectedly, using a transcriptional terminator insertion strategy, we found that disrupting the Malat1 gene without altering the expression of its adjacent genes in a transgenic mouse model of breast cancer drastically promoted lung metastasis, and importantly, this phenotype was completely reversed by genetic add-back of Malat1. Moreover, CRISPR-Cas9- mediated knockout of MALAT1 in human breast cancer cells induced their metastatic ability, which was reversed by Malat1 re-expression. Conversely, overexpression of Malat1 suppressed breast cancer metastasis in both transgenic mice and xenograft models. Mechanistically, we used a recently developed chromatin isolation by RNA purification-mass spectrometry (ChIRP-MS) approach to identify TEAD family members as binding proteins for MALAT1 at the endogenous level from primary mammary tumor tissues, and discovered that MALAT1 binds, sequesters, and inactivates the pro-metastatic transcription factor TEAD. We also found an inverse correlation of MALAT1 levels with breast cancer progression and metastasis. Based on these important findings, we propose to comprehensively characterize the loss-of-function and gain-of-function effects of MALAT1 in breast cancer metastasis, using genetically engineered mouse models, transplantation models, syngeneic models, xenograft models, and CRISPR-Cas9 genome editing approaches (Specific Aim 1); we will also elucidate the mechanism by which MALAT1 regulates metastasis (Specific Aim 2). This project will lead to a major revision of the current model for a highly abundant and conserved lncRNA, and will profoundly advance the understanding of lncRNA's functions and mechanisms of action in tumor metastasis.