南方医科大学学报

南方医科大学学报 ›› 2018, Vol. 38 ›› Issue (05): 505-.

• •    下一篇

MircoRNA-218下调BMI-1表达水平介导的抗骨肉瘤作用

赖桂华，黄爱兰，赵志，卢兴浩，祖文轩   

  • 出版日期:2018-05-20 发布日期:2018-05-20

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

  • Online:2018-05-20 Published:2018-05-20

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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。

Abstract: 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-218was 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 for 24, 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.