骨肉瘤干细胞差异表达microRNA对其干性特性的影响  

The aberrant expression of microRNA and its influence on biological characteristics of osteosarcoma stem cells

作　　者：王芊[1] 张博文 罗鹏 郭建勋[1] 王宏刚[1] 聂井君 王任先[1] 陈大福[1] WANG Qian;ZHANG Bo-wen;LUO Peng;GUO Jian-xun;WANG Hong-gang;NIE Jing-jun;WANG Ren-xian;CHEN Da-fu(Laboratory of Bone Tissue Engineering,Beijing Institute of Traumatology and Orthopaedics,Beijing Jishuitan Hospital,Beijing 100035,China)

机构地区：[1]北京市创伤骨科研究所骨组织工程研究室/北京积水潭医院,100035

出　　处：《中国医药生物技术》2022年第3期214-219,共6页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金面上项目(51973021、52173275);北京市属医学科研院所公益发展改革试点项目(第四批:京医研2021-6)。

摘　　要：目的 探讨骨肉瘤干细胞差异表达microRNA(miRNA)及其对骨肉瘤干细胞的干性特性(自我更新、侵袭、凋亡)的影响。方法 采用无血清悬浮培养法和流式分选术分离鉴定骨肉瘤干细胞;分别采用成球实验、体外侵袭鉴定骨肉瘤干细胞的干性特性;高通量测序检测骨肉瘤干细胞中差异表达miRNA;采用定量逆转录PCR(qRT-PCR)技术检测相关差异表达miRNA的情况,验证miRNA结果;相关miRNA抑制剂干扰后,分别进行成球实验和侵袭实验检测miRNA对骨肉瘤干细胞自我更新、侵袭能力和细胞凋亡的影响。结果 成功分离Saos-2的CD133^(+)和CD133^(+)细胞,具有较强的自我更新能力和侵袭能力。与CD133^(+)细胞相比,CD133^(+)细胞的高表达miRNAs共有15个;低表达miRNAs共有2个。采用qPCR技术验证高通量测序差异表达miRNA相关结果,CD133^(+)亚群中miR-33b-3p、miR-3648、miR-4442、miR-5091表达上调,miR-3143、miR-3150a-5p表达下调。抑制相关高表达miRNA(miR-33b-3p、miR-3648、miR-4442、miR-5091)表达后,CD133^(+)细胞的自我更新能力和侵袭均下降,细胞凋亡上升。结论 骨肉瘤干细胞中具有多种差异表达miRNA,其对骨肉瘤干细胞的干性特性具有调控作用。Objective We aim to investigate the aberrant expression of microRNA(miRNA) in osteosarcoma stem cells(OSCs) and their effects on biological characteristics of OSCs.Methods The serum-free suspension culture and flow cytometry sorting technology were used to isolate human OSCs. Biological characteristics of OSCs were evaluated by sphere colony formation assay, invasion assay and apoptosis assay in vitro. miRNA microarray was performed to analyze differentially expressed mi RNA in OSCs. Quantitative RT-PCR was used to validate the microarray data of selected miRNA. The effect of miRNA inhibitors on biological characteristics of OSCs were examined.Results CD133^(+)and CD133^(+)cells from Saos-2 cell line were successfully isolated. Functional studies revealed that CD133^(+)cells from Saos-2 cell line had the characteristics of cancer stem-like cell, which formed more sphere colonies, showed higher invasiveness than CD133^(+)cells. Compared with CD133^(+)cells, 15 highly expressed miRNA and 2 lowly expressed miRNA were identified by miRNA array in CD133^(+)cells. qRT-PCR analysis validated that miR-33b-3p, miR-3648, miR-4442, miR-5091 were up-regulated, and miR-3143 and miR-3150a-5p were down-regulated in CD133^(+)cells from Saos-2 cell line. Inhibition of the up-regulated miRNA(miR-33b-3p, miR-3648, miR-4442, miR-5091) could suppress the self-renewal capacity, invasion ability and enhance apoptosis in CD133^(+)cells from Saos-2 cell line.Conclusion Several differentially expressed miRNA in human OSCs could regulate biological characteristics of OSCs.

关 键 词：骨肉瘤干细胞 CD133 自我更新 侵袭 

分 类 号：R738.1[医药卫生—肿瘤]