淋巴结（LN）转移是非小细胞肺癌（NSCLC）的主要转移途径之一，被认为是患者预后不良的主要原因。尽管淋巴管生成被广泛认为是介导淋巴结转移的关键过程，但非小细胞肺癌中涉及淋巴管生成和淋巴结转移的调节机制仍不清楚。在这项研究中，我们采用高通量测序来鉴定一种新型环状 RNA (circRNA)，circTLCD4-RWDD3，它在 LN 转移性 NSCLC 的细胞外囊泡 (EV) 中显着上调，并且与 NSCLC 患者的 OS 和 DFS 恶化呈正相关。来自多中心临床队列的 NSCLC。下调 EV 包装的 circTLCD4-RWDD3 的表达可在体外和体内抑制 NSCLC 的淋巴管生成和淋巴结转移。从机械角度来看，circTLCD4-RWDD3 与 hnRNPA2B1 发生物理相互作用，并通过上调 UBC9 介导 hnRNPA2B1 K108 残基处的 SUMO2 修饰。随后，circTLCD4-RWDD3诱导的SUMO化hnRNPA2B1被ALIX的SUMO相互作用基序（SIM）识别，并激活ALIX以招募ESCRT-III，从而促进将circTLCD4-RWDD3分选到NSCLC细胞来源的EV中。此外，EV包装的circTLCD4-RWDD3被淋巴内皮细胞内化，激活PROX1的转录，导致NSCLC的淋巴管生成和淋巴结转移。重要的是，通过突变 ALIX 中的 SIM 或 hnRNPA2B1 的 K108 残基来阻断 EV 介导的 circTLCD4-RWDD3 传输，可抑制体内 NSCLC 的淋巴管生成和淋巴结转移。我们的研究结果揭示了 SUMO 化 hnRNPA2B1 诱导的 circTLCD4-RWDD3 EV 包装促进 NSCLC 淋巴结转移的精确机制，表明 EV 包装的 circTLCD4-RWDD3 可能成为针对 LN 转移性 NSCLC 的潜在治疗靶点。© 2023。 ）。

Lymph node (LN) metastasis is one of the predominant metastatic routes of non-small cell lung cancer (NSCLC) and is considered as a leading cause for the unsatisfactory prognosis of patients. Although lymphangiogenesis is well-recognized as a crucial process in mediating LN metastasis, the regulatory mechanism involving lymphangiogenesis and LN metastasis in NSCLC remains unclear. In this study, we employed high-throughput sequencing to identify a novel circular RNA (circRNA), circTLCD4-RWDD3, which was significantly upregulated in extracellular vesicles (EVs) from LN metastatic NSCLC and was positively associated with deteriorated OS and DFS of patients with NSCLC from multicenter clinical cohort. Downregulating the expression of EV-packaged circTLCD4-RWDD3 inhibited lymphangiogenesis and LN metastasis of NSCLC both in vitro and in vivo. Mechanically, circTLCD4-RWDD3 physically interacted with hnRNPA2B1 and mediated the SUMO2 modification at K108 residue of hnRNPA2B1 by upregulating UBC9. Subsequently, circTLCD4-RWDD3-induced SUMOylated hnRNPA2B1 was recognized by the SUMO interaction motif (SIM) of ALIX and activated ALIX to recruit ESCRT-III, thereby facilitating the sorting of circTLCD4-RWDD3 into NSCLC cell-derived EVs. Moreover, EV-packaged circTLCD4-RWDD3 was internalized by lymphatic endothelial cells to activate the transcription of PROX1, resulting in the lymphangiogenesis and LN metastasis of NSCLC. Importantly, blocking EV-mediated transmission of circTLCD4-RWDD3 via mutating SIM in ALIX or K108 residue of hnRNPA2B1 inhibited the lymphangiogenesis and LN metastasis of NSCLC in vivo. Our findings reveal a precise mechanism underlying SUMOylated hnRNPA2B1-induced EV packaging of circTLCD4-RWDD3 in facilitating LN metastasis of NSCLC, suggesting that EV-packaged circTLCD4-RWDD3 could be a potential therapeutic target against LN metastatic NSCLC.© 2023. The Author(s).