PO.ET06.04 · 实验与分子治疗
虫草素通过靶向NAT10介导的VCAN的ac4C RNA修饰抑制肝内胆管癌进展
Cordycepin inhibits Intrahepatic cholangiocarcinoma progression by targeting NAT10-mediated ac4C RNA modification of VCAN
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：肝内胆管癌（ICC）是一种预后不良的侵袭性恶性肿瘤。在驱动肿瘤进展的RNA修饰中，由NAT10介导的N4-乙酰胞苷（ac4C）对RNA稳定性和翻译至关重要。虫草素是一种来自冬虫夏草的生物活性化合物，具有已知的抗肿瘤活性，但其通过RNA修饰通路对ICC的影响尚不清楚。本研究探讨虫草素如何靶向NAT10-ac4C轴以抑制ICC，为基于RNA修饰的治疗提供机制支持。
方法：使用CCK-8实验定量虫草素对HUCCT1和RBE细胞的抗增殖作用，以确定IC50值。采用western blot分析RNA修饰酶谱，并鉴定ac4C/NAT10为潜在靶点。通过CCK-8、EdU掺入、集落形成、流式细胞术、Transwell迁移和侵袭以及划痕愈合实验，系统评估虫草素对细胞表型的影响。通过分子对接、细胞热位移实验（CETSA）和药物亲和反应靶点稳定性（DARTS）分析验证虫草素-NAT10的直接相互作用。通过点杂交检测全局ac4C水平。采用RNA-seq结合acRIP-seq确定下游ac4C调控基因。在C57BL/6小鼠皮下ICC异种移植模型中进一步评估抗肿瘤疗效。
结果：虫草素在HUCCT1细胞中的IC50值为67.18 µM，在RBE细胞中为75.50 µM。它显著抑制增殖、迁移和侵袭，同时以剂量依赖方式诱导凋亡（P < 0.05）。虫草素显著降低NAT10蛋白丰度和全局ac4C修饰水平（P < 0.05）。对接分析预测结合能为−7.0 kcal/mol，CETSA与DARTS共同证实了直接的靶点结合。RNA-seq和acRIP-seq整合分析鉴定出细胞外基质相关基因VCAN为主要的ac4C调控下游效应因子。虫草素降低VCAN的ac4C富集和表达，而NAT10过表达逆转了这些效应。在体内，虫草素显著抑制肿瘤生长，表现为肿瘤体积和重量减少（P < 0.05）。相应地，肿瘤组织中NAT10和VCAN的表达显著下调。
结论：本研究证明虫草素直接靶向NAT10，减弱其表达和全局ac4C修饰，从而抑制下游致癌效应因子VCAN并遏制ICC进展。这些发现突显了NAT10-ac4C轴在虫草素抗肿瘤活性中的核心作用，并将该通路确定为ICC中一个有前景的治疗靶点。
查看英文原文 English abstract
Background: Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis. Among RNA modifications that drive tumor progression, N4-acetylcytidine (ac4C) mediated by NAT10 is crucial for RNA stability and translation. Cordycepin, a bioactive compound from Cordyceps sinensis, has known antitumor activity, yet its impact on ICC via RNA-modification pathways remains unclear. This study examines how cordycepin targets the NAT10-ac4C axis to inhibit ICC, providing mechanistic support for RNA-modification-based therapy.
Methods: The antiproliferative effects of cordycepin on HUCCT1 and RBE cells were quantified using CCK-8 assays to determine IC 50 values. Western blotting was employed to profile RNA-modifying enzymes and identify ac4C/NAT10 as potential targets. The effects of cordycepin on cellular phenotypes were systematically assessed via CCK-8, EdU incorporation, colony formation, flow cytometry, Transwell migration and invasion, and wound-healing assays. Direct cordycepin-NAT10 interaction was validated by molecular docking, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) analysis. Global ac4C levels were examined by dot blotting. RNA-seq combined with acRIP-seq was used to define downstream ac4C-regulated genes. Antitumor efficacy was further evaluated in C57BL/6 mouse subcutaneous ICC xenograft models.
Results: Cordycepin exhibited IC 50 values of 67.18 µM in HUCCT1 cells and 75.50 µM in RBE cells. It markedly suppressed proliferation, migration, and invasion while inducing apoptosis in a dose-dependent manner (P < 0.05). Cordycepin significantly reduced NAT10 protein abundance and global ac4C modification levels (P < 0.05). Docking analysis predicted a binding energy of −7.0 kcal/mol, and CETSA together with DARTS confirmed direct target engagement. Integrative RNA-seq and acRIP-seq analyses identified VCAN, an extracellular matrix-associated gene, as a principal ac4C-regulated downstream effector. Cordycepin decreased both ac4C enrichment and expression of VCAN, whereas NAT10 overexpression reversed these effects. In vivo, cordycepin substantially suppressed tumor growth, reflected by reduced tumor volume and weight (P < 0.05). Correspondingly, NAT10 and VCAN expression in tumor tissues were significantly downregulated.
Conclusion: This study demonstrates that cordycepin directly targets NAT10, attenuating its expression and global ac4C modification, thereby suppressing the downstream oncogenic effector VCAN and restraining ICC progression. These findings underscore the central role of the NAT10-ac4C axis in cordycepin's antitumor activity and identify this pathway as a promising therapeutic target in ICC.
利益披露 Disclosure
Y. Zhao, None..
Y. Chen, None..
Y. Fang, None..
K. Luo, None..
J. Qu, None..
D. Shang, None..
C. Xu, None..
G. Zhang, None.