ROC1 通过调控 p-IκBα/NF-κB 信号通路促进膀胱癌的恶性进展。

BACKGROUND: Regulator of cullins 1 (ROC1) is an important catalytic subunit of cullin-RING E3 ligase. Nuclear factor-kappa B (NF-κB) signaling is closely related to tumor invasion and metastasis. Earlier, we reported that ROC1 was associated with a poor prognosis in patients with bladder cancer (BCa). However, it is unclear whether ROC1 is involved in the NF-κB signaling associated with malignant BCa progression. METHODS: The expression of ROC1 and p65 in bladder cancer and paracancerous tissues were detected by immunohistochemistry (IHC). Pearson correlation was used to assess correlation between ROC1 and p65 protein expressions. The wound-healing and transwell assays were used to monitor cell invasion and migration. The effect of ROC1 on the expression of key proteins in the NF-κB signaling was determined by immunofluorescence and western blot (WB). Cycloheximide (CHX), MG132 and immunoprecipitation assays were used to evaluate the effect of ROC1 on the ubiquitination of phosphorylated inhibitor of kappa B alpha (p-IκBα). A lung metastasis mouse model was generated to detect the role of ROC1 in tumor metastasis. RESULTS: We found that ROC1 was up-regulated in BCa tissues and cell lines, and high ROC1 levels were positively correlated with higher tumour grade, lymph node metastasis, distant metastasis and poor prognosis. Linear-regression analysis showed significant a Pearson correlation between ROC1 and nuclear p65 expression in BCa tissue microarray (TMA) samples. Functional studies demonstrated that ROC1 promoted BCa cell invasion and migration. In vitro and in vivo experiments showed that ROC1 activated NF-κB signaling by enhancing the ubiquitination of p-IκBα, which caused p65 nuclear translocation and promoted the transcription of some metastasis-related target genes, such as urokinase-type plasminogen activator receptor (uPAR), intracellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and matrix metalloproteinase 9 (MMP9), resulting in promoting BCa metastasis. CONCLUSION: ROC1 plays an important role in the progression of BCa and serves as a potential diagnostic and therapeutic target for patients with BCa.

背景：环指蛋白 1（ROC1）是泛素连接酶的重要催化亚基。核因子-κB（NF-κB）信号通路与肿瘤的侵袭和转移密切相关。我们之前的研究报告显示，ROC1 与膀胱癌（BCa）患者的不良预后相关。然而，ROC1 是否参与与恶性 BCa 进展相关的 NF-κB 信号通路尚不清楚。

方法：采用免疫组织化学（IHC）检测膀胱癌及癌旁组织中 ROC1 和 p65 的表达。采用 Pearson 相关性分析评估 ROC1 蛋白表达与 p65 蛋白表达之间的相关性。采用划痕愈合实验和 Transwell 实验检测细胞侵袭和迁移。采用免疫荧光和 Western blot（WB）检测 ROC1 对 NF-κB 信号通路中关键蛋白表达的影响。采用环己酰亚胺（CHX）、MG132 和免疫沉淀实验评估 ROC1 对磷酸化 IκBα（p-IκBα）泛素化的影响。建立肺转移小鼠模型检测 ROC1 在肿瘤转移中的作用。

结果：我们发现 ROC1 在 BCa 组织和细胞系中上调，且 ROC1 高表达与肿瘤分级较高、淋巴结转移、远处转移和不良预后呈正相关。BCa 组织微阵列（TMA）样本的线性回归分析显示 ROC1 与核 p65 表达之间存在显著的 Pearson 相关性。功能研究表明，ROC1 通过增强 p-IκBα 的泛素化来促进 BCa 细胞的侵袭和迁移。体外和体内实验表明，ROC1 通过激活 NF-κB 信号通路，促进 p65 核转位，促进一些与转移相关的靶基因的转录，如尿激酶型纤溶酶原激活物受体（uPAR）、细胞间黏附分子 1（ICAM1）、血管细胞黏附分子 1（VCAM1）和基质金属蛋白酶 9（MMP9），从而促进 BCa 转移。

结论：ROC1 在 BCa 的进展中发挥重要作用，可能成为 BCa 患者的潜在诊断和治疗靶点。