Non-coding RNA functions in tumor metastasis

尽管在抗击癌症方面取得了很大进展，特别是在癌症的早期阶段，但转移 这仍然是一项艰巨的、往往是致命的挑战。很明显，非编码RNA，包括 MicroRNAs和Long Non-Coding RNAs(LncRNAs)是调控转移的分子网络的组成部分。 已知一些lncRNAs具有与其基因组位置相反的功能；例如，相反的表型 已经报道了来自lncRNA困扰的基因缺失和插入失活，并且有趣的是， 鬼魂基因的缺失是由于基因组DNA的丢失，而不是鬼魂RNA的丢失。因此，一名少校 在lncRNA研究中的挑战是，由于缺失或失活lncRNA基因而产生的表型是否可以 明确地将其归因于lncRNA本身的丧失或重叠调控的丧失 元素。MALAT1(转移相关肺腺癌转录本1)是最丰富和 在正常组织中保守的lncRNAs，以前被描述为肿瘤转移的促进剂。然而， 没有证据表明之前报道的MALAT1基因缺失(导致多发性硬化 MALAT1‘S邻近基因)或反义核糖核酸(从未通过救援实验或通过 MALAT1基因敲除细胞)的效应是MALAT1基因缺失所特有的。出乎意料的是，使用转录的 终止子插入策略，我们发现在不改变其表达的情况下干扰MALAT1基因 乳腺癌转基因小鼠模型中的相邻基因极大地促进了肺转移， 重要的是，这种表型被MALAT1的基因加回完全逆转。此外，CRISPR-CAS9- MALAT1在人乳腺癌细胞中的介导敲除诱导其转移能力，而这种转移能力被逆转 通过MALAT1的重新表达。相反，MALAT1的过表达抑制了两者的乳腺癌转移 转基因小鼠和异种移植模型。从机制上讲，我们使用了一种最新开发的染色质分离方法 RNA纯化-质谱法鉴定TEAD家族成员为结合蛋白 从原发乳腺癌组织的内源性水平上结合MALAT1，并发现MALAT1结合， 隔离，并使促进转移的转录因子tead失活。我们还发现了一个负相关关系 MALAT1水平与乳腺癌进展和转移的关系。基于这些重要发现，我们 建议综合表征MALAT1在乳房中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、同基因模型、 异种移植模型和CRISPR-Cas9基因组编辑方法(特定目标1)；我们还将阐明 MALAT1调节转移的机制(特定目标2)。这个项目将导致对 目前的模型为高度丰富和保守的lncRNA，并将深刻地推动对 研究了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.