miR-335靶向Sp1对人骨肉瘤MG-63细胞增殖的调控

miR-335 regulates proliferation of human osteosarcoma MG-63 cells by targeting Sp1

目的:探讨miR-335对人骨肉瘤MG-63细胞增殖的调控是否通过靶向抑制Sp1基因的表达实现。方法:利用萤光素酶报告基因实验验证miR-335能否靶向作用于Sp1基因的3’-非翻译区(untranslated region,UTR);利用real-time PCR和Western blotting分析Sp1 mRNA和蛋白表达的变化;通过MTT法分析MG-63细胞增殖水平。结果:萤光素酶报告基因实验结果显示,miR-335可特异性结合于Sp1基因的3’-UTR。Real-time PCR和Western blotting结果显示,转染miR-335可减少Sp1蛋白的表达,但对mRNA表达无显著影响;转染Sp1表达质粒可上调Sp1 mRNA和蛋白表达。MTT结果显示,miR-335可抑制MG-63细胞增殖,转染Sp1表达质粒可部分逆转miR-335对MG-63细胞增殖的抑制作用。结论:miR-335可能通过靶向Sp1基因抑制人骨肉瘤MG-63细胞增殖。

AIM： To investigate the regulatory role of miR-335 on the proliferation of human osteosarcoma cell line MG-63. METHODS ： Luciferase reporter gene assay was used to detect the specific binding ability of miR-335 to Spl 3＇-untranslated region （UTR）. Real-time PCR and Western blotting were used to evaluate the expression of Spl mRNA and protein, respectively. MTr assay was used to analyze the proliferation of MG-63 cells. RESULTS： The luciferase re- porter gene assay showed that miR-335 targeted Spl 3＇ -UTR. Western blotting and real-time PCR showed that miR-335 in- hibited the protein expression of Spl, but had no effect on the mRNA expression of Spl. Transfection of Spl expression plasmid increased the protein.and mRNA expression of Spl. MTr assay showed the viability of MG-63 cells transfected with miR-335 was significantly decreased compared with negative control. Transfection of Spl expression plasmid partly reversed the inhibitory effect of miR-335 on the proliferation of MG-63 cells. CONCLUSION： miR-335 inhibits the proliferation of human osteosarcoma MG-63 cells, which may be related to its targeting on Spl 3＇ -UTR and the subsequent down-regulation of Spl expression. [