A long noncoding RNA antagonizes the PBAF complex to promote ccRCC progression

项目摘要：我们发现了一种完全没有特征的长非编码RNA(LncRNA)ADIRF- AS1与肿瘤抑制因子多溴和BRG1相关因子(PBAF)染色质结合 修饰复合体，包括PBAF复合体的三个独特的蛋白质成分：ARID2、BRD7和 PBRM1。含有PBRM1蛋白的PBAF复合溴域在大约40%的Clear 肾细胞癌(CcRCC)，其中PBRM1的缺失增加了增殖和总体肿瘤负担。公开地 现有的患者资料显示ADIRF-AS1在肾细胞癌组织中的表达高于正常组织。 纸巾。我们在一组肾细胞癌细胞系中过表达ADIRF-AS1，并发现ADIRF-AS1过表达 仅在野生型表达PBAF蛋白的ccRCC细胞系中显著促进增殖 组件。重要的是，我们发现ADIRF-AS1的过表达降低了PBAF的蛋白表达 蛋白质成分。接下来，我们试图确定ADIRF-AS1是如何负性调节PBAF复合体的。我们 进行了lncRNA下拉试验，然后进行了蛋白质组学分析，发现与 PBAF复合体ADIRF-AS1还与E3泛素连接酶TRIM25结合，已被证明 靶向蛋白酶体降解的PBRM1。我们验证了沉默TRIM25会增加PBRM1的表达 并发现蛋白酶体抑制剂挽救了ADIRF-AS1后PBRM1表达的减少 过度表达。因此，我推测ADIRF-AS1与PBAF复合体相互作用，促进其 通过作为PBRM1和TRIM25的支架进行降解，促进肿瘤的形成和进展。 为了解决这一假设，我们提出了两个目标：(1)确定ADIRF-AS1在PBAF中的作用 介导的代谢和肿瘤发生，以及(2)表征ADIRF-AS1负性的机制 通过TRIM25调节PBAF复合体，促进肿瘤发生。

Project Summary: We discovered that a completely uncharacterized long noncoding RNA (lncRNA) ADIRF- AS1 binds to the tumor suppressor Polybromo- and BRG1-associated factors-containing (PBAF) chromatin modifying complex, including the three unique protein components of the PBAF complex: ARID2, BRD7, and PBRM1. The PBAF complex bromodomain containing protein PBRM1 is mutated in approximately 40% of clear cell renal carcinoma (ccRCC), where loss of PBRM1 increases proliferation and overall tumor burden. Publicly available patient data revealed that ADIRF-AS1 is more highly expressed in ccRCC tumors compared to normal tissues. We overexpressed ADIRF-AS1 in a panel of ccRCC cell lines and found that ADIRF-AS1 overexpression significantly promoted proliferation only in ccRCC cell lines with wildtype expression of PBAF protein components. Importantly, we found that overexpression of ADIRF-AS1 decreased protein expression of PBAF protein components. Next, we sought to identify how ADIRF-AS1 negatively regulates the PBAF complex. We performed a lncRNA pulldown assay followed by proteomics analysis and found that along with components of the PBAF complex, ADIRF-AS1 also associates with an E3 ubiquitin ligase TRIM25, which has been shown to target PBRM1 for proteasomal degradation. We validated that silencing TRIM25 increases PBRM1 expression and found that proteasome inhibitors rescued decreased expression of PBRM1 after ADIRF-AS1 overexpression. Therefore, I hypothesize that ADIRF-AS1 interacts with the PBAF complex to promote its degradation by acting as a scaffold for PBRM1 and TRIM25, promoting tumor formation and progression. To address this hypothesis, we propose two aims to: (1) Determine the role of ADIRF-AS1 expression in PBAF mediated metabolism and tumorigenesis, and (2) Characterize the mechanism by which ADIRF-AS1 negatively regulates the PBAF complex through TRIM25 to promote tumorigenesis.