Li Ran, Ruan Qing, Zheng Jia, Zhang Butian, Yang Hongliang
Department of Orthopedics, China-Japan Union Hospital of Jilin University, Changchun, China.
Department of Radiology, China-Japan Union Hospital of Jilin University, Changchun, China.
Front Pharmacol. 2021 Mar 8;12:632206. doi: 10.3389/fphar.2021.632206. eCollection 2021.
Development of resistance to doxorubicin-based chemotherapy limits its curative effect in osteosarcoma. In the current study, we focused on investigating the mechanisms underlying the development of doxorubicin resistance in osteosarcoma. The human osteosarcoma cell line MG-63 and doxorubicin-resistant MG-63/Dox cells were used in this study. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of the long non-coding RNA LINC01116 in the two cell lines. Then, the specific shRNA for LINC01116 was employed to suppress LINC01116 expression in MG-63/Dox cells. Cell viability was assessed by the CCK-8 and colony formation assays. Cell migration and invasion were evaluated by the transwell assay. Moreover, the epithelial-mesenchymal transition (EMT)-related proteins, E-cadherin, vimentin, and N-cadherin were evaluated by Western blotting. The regulation of LINC01116 on miR-424-5p expression was examined using methylation-specific PCR, RNA immunoprecipitation, and Western blotting assay. The potential targeting of HMGA2 by miR-424-5p was predicted using the bioinformatics databases TargetScan and miRanda and verified by a dual-luciferase reporter assay. LINC01116 was more highly expressed in MG-63/Dox cells than in MG-63 cells. Inhibition of LINC01116 suppressed cell viability, migration, and invasion, along with upregulating the expression of E-cadherin, downregulating vimentin, and attenuating doxorubicin resistance in MG-63/Dox cells. Further mechanism-related investigations indicated that LINC01116 regulated HMGA2 expression via the EZH2-associated silencing of miR-424-5p. LINC01116 exerts regulatory effects on doxorubicin resistance through the miR-424-5p axis, providing a potential approach to overcoming chemoresistance in osteosarcoma.
对基于阿霉素的化疗产生耐药性限制了其在骨肉瘤中的治疗效果。在本研究中，我们着重探究骨肉瘤中阿霉素耐药性产生的潜在机制。本研究使用了人骨肉瘤细胞系MG-63和阿霉素耐药的MG-63/Dox细胞。采用定量实时聚合酶链反应（qRT-PCR）检测两种细胞系中长链非编码RNA LINC01116的表达。随后，利用针对LINC01116的特异性短发夹RNA（shRNA）抑制MG-63/Dox细胞中LINC01116的表达。通过CCK-8和集落形成试验评估细胞活力。采用Transwell试验评估细胞迁移和侵袭能力。此外，通过蛋白质免疫印迹法评估上皮-间质转化（EMT）相关蛋白E-钙黏蛋白、波形蛋白和N-钙黏蛋白。利用甲基化特异性PCR、RNA免疫沉淀和蛋白质免疫印迹试验检测LINC01116对miR-424-5p表达的调控作用。使用生物信息学数据库TargetScan和miRanda预测miR-424-5p对HMGA2的潜在靶向作用，并通过双荧光素酶报告基因试验进行验证。LINC01116在MG-63/Dox细胞中的表达高于MG-63细胞。抑制LINC01116可抑制MG-63/Dox细胞的活力、迁移和侵袭，同时上调E-钙黏蛋白的表达，下调波形蛋白的表达，并减弱其对阿霉素的耐药性。进一步的机制研究表明，LINC01116通过EZH2相关的miR-424-5p沉默来调控HMGA2的表达。LINC01116通过miR-424-5p轴对阿霉素耐药性发挥调控作用，为克服骨肉瘤化疗耐药性提供了一种潜在方法。
