摘要
目的:探讨miR-29a-3p是否影响骨肉瘤细胞干性并探究其分子机制。方法:通过低黏附培养法富集骨肉瘤干细胞,利用real-time PCR实验检测骨肉瘤干细胞分子标志证实干细胞富集成功,并分别检测miR-29a-3p在骨肉瘤细胞系以及干细胞中的表达。而后通过调控miR-29a-3p表达检测其对骨肉瘤干细胞分子标志(Nanog、Oct-4、Sox2)表达水平以及克隆形成能力的影响。利用生物信息学网站TargetScan预测得到miR-29a-3p的靶基因,即肿瘤坏死因子受体相关因子5(tumor necrosis factor receptor associated factor-5,TRAF5),而后通过双荧光素酶报告基因实验证实miR-29a-3p与TRAF5的靶向关系,并利用real-time PCR、Western blotting实验检测miR-29a-3p对TRAF5表达的影响。通过拯救实验证实miR-29a-3p通过抑制TRAF5调控骨肉瘤细胞干性分子标志表达水平以及克隆形成能力。为了进一步探究miR-29a-3p发挥上述功能与Wnt通路的关系,我们检测了Wnt信号通路中β-catenin的入核情况,并利用TOPFlash报告基因质粒检测β-catenin介导的TCF/LEF转录活性水平从而指征Wnt通路的激活情况,最后通过Western blotting检测Wnt信号通路相关蛋白TCF-4、Pygo2的表达水平。结果:real-time PCR实验结果表明miR-29a-3p在骨肉瘤细胞系以及干细胞中异常低表达,随后发现miR-29a-3p能够抑制骨肉瘤干细胞分子标志表达水平以及克隆形成能力。生物信息学分析、分子生物学实验以及拯救实验结果表明,miR-29a-3p通过靶向TRAF5发挥上述功能。进一步的研究发现miR-29a-3p下调能够促进Wnt信号通路中β-catenin入核、TCF/LEF转录活性水平以及Wnt信号通路相关蛋白TCF-4、Pygo2的表达水平,而同时下调miR-29a-3p、TRAF5表达可以部分逆转上述现象。结论:miR-29a-3p通过靶向TRAF5影响Wnt信号通路继而调控骨肉瘤细胞干性。
Objective:To study whether miR-29a-3p affects osteosarcoma cells stemness and explore its underlying molecular mechanism.Methods:Osteosarcoma stem cells were enriched and cultured on ultralow attachment Corning plates and grown in sphere media,real-time PCR assays were used to detect the level of miR-29a-3p in osteosarcoma cell lines and stem cells.Then,by regulating the expression of miR-29a-3p,its effects on the expression levels of osteosarcoma steMness markers,such as Nanog,Oct-4,Sox2,and clone formation capacities were detected.Online bioinformatics prediction through TargetScan website was performed to testify the targeting gene of miR-29a-3p,which was TRAF5,tumor necrosis factor receptor associated factor-5.Then,the targeting relationship between miR-29a-3p and TRAF5 was confirmed through dual luciferase reporter assays.And we also explored the effects of miR-29a-3p on TRAF5 expression on mRNA or protein level by real-time PCR,Western blotting experiments.Rescue experiments were also performed to verify that miR-29a-3p can regulate osteosarcoma stemness markers expression and clone formation capacities by targeting TRAF5.To further investigate whether Wnt/β-catenin pathway participates in the above mentioned roles of miR-29a-3p,we inspected the nuclear accumulation of β-catenin and used TOPFlash reporter plasmids to examine the activation level of Wnt signal pathway.Finally,we detected the expression level of Wnt-related proteins,TCF-4,Pygo2 by Western blotting.Results:Real-time PCR results showed that miR-29a-3p was aberrantly downregulated in osteosarcoma cell lines and stem cells,and we also found miR-29a-3p could inhibit stemness markers expression and clone formation capacities of osteosarcoma stem cells.Bioinformatics prediction and molecular biology experiments demonstrated that miR-29a-3p could inhibit osteosarcoma stem cells stemness through targeting TRAF5.Further studies showed miR-29a-3p inhibition could promote nuclear accumulation of β-catenin,TCF/LEF transcription level and upregulation of Wnt-related proteins,TCF-4,Pygo2.However,simultaneously inhibition of miR-29a-3p and TRAF5 could partly reverse the above phenomenon.Conclusion:miR-29a-3p could repress the stemness of osteosarcoma stem cells through Wnt/β-catenin pathway by targeting TRAF5.
作者
匡健
董文刚
丁家云
刘炅
刘文毅
周志坚
刘军
KUANG Jian;DONG Wengang;DING Jiayun;LIU Jiong;LIU Wenyi;ZHOU Zhijian;LIU Jun(Southern Center for Diseases Control and Prevention,Guangdong Guangzhou 510630,China;Department of Emergency Surgery,Shaanxi Provincial People's Hospital,Shaanxi Xi'an 710068,China)
出处
《现代肿瘤医学》
2025年第3期388-395,共8页
Journal of Modern Oncology
基金
广东省基础与应用基础研究基金资助项目(编号:2020A1515010299,2020A1515010931)。
作者简介
匡健(1993-),男,江西吉安人,助理工程师,主要从事肿瘤基础研究。E-mail:413846165@qq.com;通信作者:刘军(1993-),男,陕西汉阴人,副研究员,主要从事骨肉瘤恶性进展相关研究。E-mail:ljnbzqcdc2018@163.com。
