Zhang Jingyong, Wang Shubao, Bai Yang, Ali Aasi Mohammad, Deng Jiewen, Chen Yushi, Fu Yonghui, He Ming
Department of Orthopedics, Shengjing Hospital of China Medical University, Shenyang 110004, China.
Department of Pathology, Shengjing Hospital of China Medical University, Shenyang 110004, China.
J Clin Med. 2023 Feb 1;12(3):1177. doi: 10.3390/jcm12031177.
First-line treatment for osteosarcoma includes chemotherapy and surgery. However, the five-year survival rate of refractory osteosarcoma remains unsatisfactory. Osteosarcoma cancer stem cells, possessing stemness and chemoresistance, are one of the critical causes of poor response to chemotherapy. Elucidating regulatory signaling pathways of osteosarcoma cancer stem cells may provide a rationale for improving regimens against chemoresistant osteosarcoma. Methotrexate (MTX)-resistant osteosarcoma cells were established. microRNA expression profiles were used for detecting differentially expressed microRNA in resistant clones and the parental cells. microRNA target databases were employed to predict potential microRNA and mRNA interactions. Flow cytometry was performed to measure stem cell marker Prominin-1 (CD133)-positive cells. Immunofluorescence staining was applied to detect CD133 expression. miR-197-3p mimic or anti-miR-197-3p stably transfected cells were used to generate xenograft models. In the study, we found that miR-197-3p was increased in MTX-resistant cell lines. Overexpression of miR-197-3p enhanced the expression of cancer stem cell markers CD133, Octamer-binding protein 4 (), Transcription factor SOX-2 (), and Homeobox protein NANOG (), as well as chemoresistance-associated genes ATP-dependent translocase ABCB1 () and Broad substrate specificity ATP-binding cassette transporter ABCG2 (), whereas miR-197-3p knockdown inhibited stemness and recovered sensitivity to MTX. We also classified the tumor suppressor Speckle-type POZ protein-like () as a target of miR-197-3p. The miR-197-3p mutation that could not combine promoter regions was unable to sustain stemness or chemoresistance. Collectively, we discovered miR-197-3p conferred osteosarcoma stemness and chemotherapy resistance by targeting , prompting promising therapeutic candidates for refractory osteosarcoma treatment.
骨肉瘤的一线治疗包括化疗和手术。然而，难治性骨肉瘤的五年生存率仍不尽人意。骨肉瘤癌干细胞具有干性和化疗耐药性，是化疗反应不佳的关键原因之一。阐明骨肉瘤癌干细胞的调控信号通路可能为改进抗化疗耐药性骨肉瘤的治疗方案提供理论依据。建立了甲氨蝶呤（MTX）耐药的骨肉瘤细胞系。利用微小RNA表达谱检测耐药克隆和亲本细胞中差异表达的微小RNA。使用微小RNA靶标数据库预测潜在的微小RNA与信使核糖核酸的相互作用。进行流式细胞术以测量干细胞标志物Prominin-1（CD133）阳性细胞。应用免疫荧光染色检测CD133表达。使用稳定转染了miR-197-3p模拟物或抗miR-197-3p的细胞来建立异种移植模型。在该研究中，我们发现miR-197-3p在MTX耐药细胞系中表达增加。miR-197-3p的过表达增强了癌干细胞标志物CD133、八聚体结合蛋白4（）、转录因子SOX-2（）和同源盒蛋白NANOG（）的表达，以及与化疗耐药相关的基因ATP依赖性转运蛋白ABCB1（）和广谱底物特异性ATP结合盒转运蛋白ABCG2（）的表达，而miR-197-3p敲低则抑制了干性并恢复了对MTX的敏感性。我们还将肿瘤抑制因子斑点型POZ蛋白样（）归类为miR-197-3p的靶标。不能结合启动子区域的miR-197-3p突变无法维持干性或化疗耐药性。总的来说，我们发现miR-197-3p通过靶向赋予骨肉瘤干性和化疗耐药性，为难治性骨肉瘤治疗提供了有前景的治疗候选方案。
