骨肉瘤是儿童和青少年中最常见的原发性骨恶性肿瘤。 Polo 样激酶 1 (PLK1) 的过度表达在骨肉瘤中很常见，可驱动疾病进展和转移，使其成为一个有前途的治疗靶点。在这项研究中，我们利用高保真 Cas13d (hfCas13d) 介导的 RNA 干扰探索骨肉瘤细胞中的 PLK1 敲低。与正常骨相比，PLK1 在骨肉瘤肿瘤组织中显着上调。通过 hfCas13d 转染 sgRNA 介导的 PLK1 抑制可抑制骨肉瘤细胞增殖，诱导 G2/M 细胞周期停滞，促进细胞凋亡，减少细胞侵袭并增加上皮标记物 E-钙粘蛋白的表达。 TurboID 的邻近标记结合免疫共沉淀确定了 PLK1 与 Smad3（TGF-β 信号传导的关键细胞内转导器）之间的新型相互作用。 PLK1 敲低会损害 Smad2/3 磷酸化并调节 TGF-β/Smad3 通路失活。最后，体内递送靶向 PLK1 的 hfCas13d 载体显着减弱了裸鼠体内骨肉瘤异种移植物的生长。总而言之，这项研究强调 PLK1 作为骨肉瘤的潜在治疗靶点和疾病进展的驱动因素。它还证明了 hfCas13d 介导的基因敲低作为靶向治疗策略的实用性。进一步优化 PLK1 抑制方法可能最终改善骨肉瘤患者的临床结果。© 2024 作者。细胞与分子医学基金会和约翰·威利出版的《细胞与分子医学杂志》

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Overexpression of polo-like kinase 1 (PLK1) is frequent in osteosarcoma and drives disease progression and metastasis, making it a promising therapeutic target. In this study, we explored PLK1 knockdown in osteosarcoma cells using RNA interference mediated by high-fidelity Cas13d (hfCas13d). PLK1 was found to be significantly upregulated in osteosarcoma tumour tissues compared to normal bone. sgRNA-mediated PLK1 suppression via hfCas13d transfection inhibited osteosarcoma cell proliferation, induced G2/M cell cycle arrest, promoted apoptosis, reduced cell invasion and increased expression of the epithelial marker E-cadherin. Proximity labelling by TurboID coupled with co-immunoprecipitation identified novel PLK1 interactions with Smad3, a key intracellular transducer of TGF-β signalling. PLK1 knockdown impaired Smad2/3 phosphorylation and modulated TGF-β/Smad3 pathway inactivation. Finally, in vivo delivery of hfCas13d vectors targeting PLK1 substantially attenuated osteosarcoma xenograft growth in nude mice. Taken together, this study highlights PLK1 as a potential therapeutic target and driver of disease progression in osteosarcoma. It also demonstrates the utility of hfCas13d-mediated gene knockdown as a strategy for targeted therapy. Further optimization of PLK1 suppression approaches may ultimately improve clinical outcomes for osteosarcoma patients.© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.