Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong, China.
Dongguan Humen Hospital, Dongguan, Guangdong, China.
J Gene Med. 2024 Oct;26(10):e3743. doi: 10.1002/jgm.3743.
Non-small cell lung cancer (NSCLC) is the main type of lung cancer with high morbidity and mortality. Vascular mimicry (VM), a distinct microcirculation model in tumors that differs from classical angiogenesis, is strongly associated with poor clinical outcomes in cancer patients. miR-491-5p has been reported to prevent NSCLC progression, including proliferation, metastasis, and angiogenesis. However, the effect and mechanism of miR-491-5p on VM have not been studied in NSCLC.
The expression of miR-491-5p was detected by quantitative reverse transcription PCR (qPCR) and fluorescence in situ hybridization (FISH). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining assays were used to examine cell growth. Tube formation assay was used to assess VM in NSCLC cells. Immunohistochemistry (IHC) and western blot were performed to detect protein expression. Immunoprecipitation was used to confirm the interaction between OTU deubiquitinase 7B (OTUD7B) and vascular endothelial growth factor A (VEGFA), and the level of ubiquitinated VEGFA. A nude mouse tumorigenesis model was used to evaluate the carcinogenic capacity of NSCLC cells in vivo. Luciferase reporter assay was used to identify the potential target of miR-491-5p.
MiR-491-5p was found downregulated in NSCLC tissues, and miR-491-5p deficiency was strongly associated with angiogenesis. miR-491-5p mimics suppressed cell viability, migration, and VM. Conversely, an inhibitor of miR-491-5p had the opposite effect. OTUD7B, a deubiquitinase, was identified as a downstream target of miR-491-5p. A luciferase reporter assay indicated that miR-491-5p directly binds to the 3'UTR of OTUD7B. Moreover, mimics of miR-491-5p caused a significant reduction in the OTUD7B protein in NSCLC cells, and an inhibitor of miR-491-5p stabilized the OTUD7B protein. In addition, overexpression of OTUD7B promoted cell proliferation, migration, and VM, similar to the effects of an inhibitor of miR-491-5p. Further exploration revealed that OTUD7B interacts with VEGFA and that the miR-491-5p-OTUD7B axis modulates the ubiquitination of VEGFA. The rescue experiment indicated that OTUD7B compromised the inhibitory effects of miR-491-5p on the cellular function of NSCLC cells.
Overall, our study first proved that miR-491-5p impedes VM by suppressing OUTD7B and promoting the ubiquitination of VEGFA. The miR-491-5p/OTUD7B axis may be a novel target for antiangiogenic therapy in NSCLC.
非小细胞肺癌（NSCLC）是一种发病率和死亡率都很高的主要肺癌类型。血管拟态（VM）是肿瘤中一种与经典血管生成不同的独特微循环模式，与癌症患者的不良临床结局密切相关。miR-491-5p 已被报道可抑制 NSCLC 的进展，包括增殖、转移和血管生成。然而，miR-491-5p 对 NSCLC 中 VM 的作用和机制尚未得到研究。
通过定量逆转录 PCR（qPCR）和荧光原位杂交（FISH）检测 miR-491-5p 的表达。使用细胞计数试剂盒-8（CCK-8）和 5-乙炔基-2'-脱氧尿苷（EdU）染色检测细胞生长。管形成实验用于评估 NSCLC 细胞中的 VM。免疫组织化学（IHC）和 Western blot 用于检测蛋白表达。免疫沉淀用于证实 OTU 去泛素酶 7B（OTUD7B）和血管内皮生长因子 A（VEGFA）之间的相互作用，以及泛素化 VEGFA 的水平。裸鼠肿瘤发生模型用于评估 NSCLC 细胞在体内的致癌能力。荧光素酶报告基因实验用于鉴定 miR-491-5p 的潜在靶标。
miR-491-5p 在 NSCLC 组织中表达下调，miR-491-5p 缺乏与血管生成密切相关。miR-491-5p 模拟物抑制细胞活力、迁移和 VM。相反，miR-491-5p 的抑制剂则产生相反的效果。OTUD7B，一种去泛素酶，被鉴定为 miR-491-5p 的下游靶标。荧光素酶报告基因实验表明，miR-491-5p 可直接结合 OTUD7B 的 3'UTR。此外，miR-491-5p 的模拟物在 NSCLC 细胞中显著降低了 OTUD7B 蛋白的表达，而 miR-491-5p 的抑制剂稳定了 OTUD7B 蛋白。此外，OTUD7B 的过表达促进了细胞增殖、迁移和 VM，类似于 miR-491-5p 抑制剂的作用。进一步探索表明，OTUD7B 与 VEGFA 相互作用，miR-491-5p-OTUD7B 轴调节 VEGFA 的泛素化。挽救实验表明，OTUD7B 削弱了 miR-491-5p 对 NSCLC 细胞细胞功能的抑制作用。
总的来说，我们的研究首次证明 miR-491-5p 通过抑制 OUTD7B 和促进 VEGFA 的泛素化来抑制 VM。miR-491-5p/OTUD7B 轴可能是 NSCLC 抗血管生成治疗的一个新靶点。
