Project 3: lncRNA SNHG1 and ATG7 in Basal-subtype Muscle-invasive Bladder Tumorigenesis

项目3：总结 这个项目的主要目标是研究构成膀胱关键步骤的分子机制。 癌症(BC)进展：侵袭和转移。具体来说，我们将重点研究自噬相关基因7是如何 (ATG7)介导的自噬信号驱动BC细胞体外侵袭和体内转移。在过去的几年里 资助期，我们发现基底亚型肌肉浸润性膀胱癌(MIBC)是由膀胱癌诱发的 致癌物N-丁基-N-(4-羟基丁基)亚硝胺(BBN)显著过度表达ATG7和长非编码 核糖核酸(LncRNA)小核仁RNA宿主基因1(SNHG1)，并显著上调自噬。在……里面 与之形成鲜明对比的是，敲击小鼠缺乏XIAP环状结构域，对BBN诱导的小鼠完全抵抗 基础MIBCs，显著减少了自噬。我们还发现，在BBN介导的膀胱中 肿瘤的发生，XIAP的环状结构域是SNHG1过表达的必要条件，而SNHG1的异位表达 SNHG1体外诱导自噬并促进BC细胞侵袭并上调ATG7、MMP2 和MMP9。此外，我们还发现ATG7基因的敲除强烈地抑制了自噬，取消了BC细胞 侵袭并降低基本MIBC标志物KRT14的表达。这些数据揭示了一个迄今未知的事实 自噬在基础MIBC形成中的作用。根据这些数据，我们假设SNHG1的上调 和ATG7通过XIAP的环区，这些分子触发的自噬信号起着至关重要的作用 在基础MIBC发生和发展中的作用。我们将在三个具体目标上检验这一假设。目标1 将定义在基础MIBC中工作的SNHG1/ATG7/自噬信号轴中的调节电路 在试管中。目的2确定SNHG1/ATG7/自噬信号在BC细胞侵袭中的生物学作用 体外和体内肿瘤的发生和转移。目标3将测试SNHG1过度表达的假设 在转基因小鼠的基础尿路上皮细胞中，促进基础MIBC的形成，而ATG7在这些细胞中的消融 基因敲除小鼠的细胞使小鼠对基本MIBC的形成和进展具有抵抗力。这些相辅相成 这些方法将为SNHG1和ATG7在体内的作用提供明确的证据。 基础MIBC的进展。而侵袭和转移是引起高死亡率的主要原因。 MIBC，人们对驱动这些至关重要的生物的主要分子或途径知之甚少 流程。我们提议的研究高度集中在一个重要但知之甚少的信号 包含SNHG1/ATG7/自噬的途径应该产生关键信息，而不仅仅是潜在的 机制，但也是新的预后生物标志物，以区分MIBC亚型和新的可用药靶点 治疗这种侵袭性的BC。

PROJECT 3: SUMMARY The major goal of this project is to study the molecular mechanisms that underlie the critical steps of bladder cancer (BC) progression: invasion and metastasis. Specifically, we will focus on how autophagy-related gene 7 (ATG7)-mediated autophagy signaling drives BC cell invasion in vitro and metastasis in vivo. During the last funding period, we found that basal-subtype muscle-invasive bladder cancer (MIBC) in mice induced by bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) markedly overexpress ATG7 and long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1), and have significantly upregulated autophagy. In stark contrast, knockin mice lacking the RING domain of XIAP, which are completely resistant to BBN-induced basal MIBCs, have markedly reduced autophagy. We also found that, during BBN-mediated bladder tumorigenesis, the RING domain of XIAP is essential for SNHG1 overexpression, and that ectopic expression of SNHG1 in vitro induces autophagy and promotes BC cell invasion accompanied by upregulated ATG7, MMP2 and MMP9. Furthermore, we showed that knockdown of ATG7 strongly inhibits autophagy, abolishes BC cell invasion and reduces the expression of basal MIBC marker KRT14. These data reveal a heretofore unknown role of autophagy in basal MIBC formation. Based on these data, we hypothesize that the upregulation of SNHG1 and ATG7 by the RING domain of XIAP, and the autophagic signaling that these molecules trigger play critical roles in the genesis and progression of basal MIBC. We will test this hypothesis in three Specific Aims. Aim 1 will define the regulatory circuitry in the SNHG1/ATG7/autophagy signaling axis that is operative in basal MIBC in vitro. Aim 2 will determine the biological effects of the SNHG1/ATG7/autophagy signaling on BC cell invasion in vitro and tumorigenesis and metastasis in vivo. Aim 3 will test the hypotheses that overexpression of SNHG1 in basal urothelial cells of transgenic mice promotes basal MIBC formation, and that ablation of ATG7 in these cells of knockout mice renders mice resistant to basal MIBC formation and progression. These complementary approaches will provide definitive evidence regarding the in vivo roles of SNHG1 and ATG7 in the formation and progression of basal MIBC. While invasion and metastasis are the main reasons of the high mortality caused by MIBC, very little is known about the principal molecules or pathways that drive these crucially important biological processes. Our proposed studies that are highly focused on an important, but poorly understood signaling pathway comprising SNHG1/ATG7/autophagy should yield critical information on not only the underlying mechanisms, but also novel prognostic biomarkers to differentiate MIBC subtypes and new druggable targets to treat this aggressive form of BC.