MircoRNA-218下调BMI-1表达水平介导的抗骨肉瘤作用（英文）  

MicroRNA-218 promotes osteosarcoma cell apoptosis by down-regulating oncogene B lymphoma mouse Moloney leukemia virus insertion region 1

作　　者：赖桂华[1] 黄爱兰 赵志[3] 卢兴浩[1] 祖文轩 LAI Guihua;HUANG Ailan;ZHAO Zhi;LU Xinghao;ZU Wenxuan(Department of Human Anatomy;Department of Chemistry, Bengbu Medical College;Department of Orthopedics, First Affiliated Hospital of Bengbu Medical College, Bengbu 233030, China)

机构地区：[1]蚌埠医学院人体解剖学教研室,安徽蚌埠233030 [2]蚌埠医学院化学教研室,安徽蚌埠233030 [3]蚌埠医学院第一附属医院骨科,安徽蚌埠233030

出　　处：《南方医科大学学报》2018年第5期505-510,共6页Journal of Southern Medical University

基　　金：Supported by Key Science Research Project Funding of Education Department of Anhui Province(KJ2017A211);General Science Research Project Funding of Education Department of Anhui Province(KJ2013Z216)~~

摘　　要：目的初步探讨microRNA-218在人骨肉瘤中的抗肿瘤作用及其分子机制。方法利用qRT-PCR检测68例人骨肉瘤组织和癌旁组织中miR-218的表达水平。将miR-218模拟物或抗miR-218模拟物转染入人骨肉瘤Saos-2细胞,通过CCK-8、Annexin V-FITC和Western blotting检测转染后的细胞活性、细胞凋亡和C-PARP蛋白表达水平。利用luciferase assay筛选和识别miR-218的作用靶点及具体作用位点,进一步通过qRT-PCR和Western blotting检测转染miR-218模拟物或抗miR-218模拟物后Saos-2细胞中BMI-1基因和蛋白的表达水平。结果人骨肉瘤组织中的miR-218表达水平明显低于癌旁组织。CCK-8结果显示,与对照组相比,转染miR-218模拟物24、36和48 h后的Saos-2细胞活性显著降低,而转染抗miR-218模拟物的Saos-2细胞活性则显著增高。同时,转染miR-218模拟物组的C-PARP的蛋白表达水平较转染抗miR-218模拟物组和对照组明显增强。Annexin V-FITC凋亡检测结果显示,转染miR-218模拟物组的细胞凋亡数和凋亡率较对照组显著增加。此外,luciferase assay结果显示Saos-2细胞中miR-218的特异性作用靶点为BMI-1,在miR-218过表达时BMI-1的基因和蛋白表达水平明显被抑制,而miR-218敲除时则显著增强;miR-218可通过直接结合BMI-1 3'-UTR抑制BMI-1的表达。结论 miR-218可能通过下调BMI-1水平促进人骨肉瘤细胞Saos-2细胞凋亡,进而发挥其抗肿瘤作用。Objective To investigate the tumor-suppressing effect of microRNA-218（miR-218） in osteosarcoma（OS） and explore its molecular mechanism. MethodsWe examined the expression levels of miR-218 in 68 pairs of OS and adjacent tissue samples using qRT-PCR. Cultured human OS cell line Saos-2 was transfected with miR-218 mimics or anti-miR-218 mimics, and the cell apoptosis was assessed using CCK-8 assay, annexin V-FITC staining andWestern blotting.We also analyzed the potential functional targets of miR-218 in Saos-2 cells using luciferase assay, qRT-PCR andWestern blotting. Results The expression level of miR-218 was lowered by at least 8 folds in OS tissues as compared with the adjacent tissues. In cultured Saos-2 cells, transfection with miR-218 mimics for24, 36, and 48 h resulted in a significant reduction in the cell viability, while transfection with anti-miR-218 mimics significantly increased the cell viability. The cells transfected with miR-218 mimics showed an obviously enhanced expression of cleaved poly（ADP-ribose） polymerase（C-PARP） as compared with the cells transfected with anti-miR-218 mimics and the control cells. Flow cytometry demonstrated obviously increased apoptosis of the cells following miR-218 mimics transfection. We identified the oncogene B lymphoma mouse Moloney leukemia virus insertion region 1（BMI-1） as a specific target of miR-218 in Saos-2 cells.BMI-1 expressions at both the mRNA and protein levels were significantly reduced in Saos-2 cells overexpressing miR-218 but increased in the cells with miR-218 knockdown as compared to the control cells. Luciferase reporter assay indicated that miR-218 directly inhibited the expression of BMI-1 via binding to its 3＇-UTR in OS cells. Conclusion miR-218 can promote OS cell apoptosis and plays the role as a tumor suppressor by down-regulating BMI-1.

关 键 词：MIR-218 BMI-1 骨肉瘤 凋亡 

分 类 号：R738.1[医药卫生—肿瘤]