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

项目3：概要 该项目的主要目标是研究膀胱癌关键步骤的分子机制， 癌症（BC）进展：侵袭和转移。具体来说，我们将重点关注自噬相关基因7 （ATG 7）介导的自噬信号传导驱动BC细胞体外侵袭和体内转移。在过去 基金期间，我们发现膀胱癌诱导的小鼠基底亚型肌层浸润性膀胱癌（MIBC） 致癌物N-丁基-N-（4-羟丁基）亚硝胺（BBN）显著过表达ATG 7和长非编码区， RNA（lncRNA）小核仁RNA宿主基因1（SNHG 1），并且具有显著上调的自噬。在 与之形成鲜明对比的是，缺乏XIAP的RING结构域的敲入小鼠，其对BBN诱导的免疫应答完全耐受。 基础MIBC具有显著减少的自噬。我们还发现，在BBN介导的膀胱 在肿瘤发生中，XIAP的RING结构域对于SNHG 1过表达是必需的， SNHG 1在体外诱导自噬并促进BC细胞侵袭，同时上调ATG 7、MMP 2 MMP9此外，我们发现ATG 7的敲低强烈抑制自噬，消除BC细胞， 并降低基础MIBC标志物KRT 14的表达。这些数据揭示了一个迄今为止未知的 自噬在基础MIBC形成中的作用。基于这些数据，我们假设SNHG 1的上调 XIAP的RING结构域与ATG 7的相互作用，以及这些分子触发的自噬信号传导起着关键作用 在基础MIBC的发生和发展中的作用。我们将在三个具体目标中检验这一假设。要求1 将定义在基础MIBC中起作用的SNHG 1/ATG 7/自噬信号传导轴中的调节回路 体外目的2：研究SNHG 1/ATG 7/自噬信号通路对BC细胞侵袭的生物学效应 体外和体内肿瘤发生和转移。目的3将验证SNHG 1过表达 在转基因小鼠基底尿路上皮细胞中ATG 7的消除促进了基底MIBC的形成， 敲除小鼠的细胞使小鼠对基础MIBC形成和进展具有抗性。这些互补 这些方法将提供关于SNHG 1和ATG 7在形成中的体内作用的明确证据， 基础MIBC进展。而肿瘤的侵袭和转移是导致恶性肿瘤高死亡率的主要原因。 MIBC，很少有人知道的主要分子或途径，驱动这些至关重要的生物 流程.我们提出的研究高度集中在一个重要的，但知之甚少的信号 包括SNHG 1/ATG 7/自噬的通路不仅应该产生关于潜在的自噬的关键信息， 机制，而且是区分MIBC亚型和新的可药用靶点的新的预后生物标志物， 治疗这种侵略性的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.