微小RNA-19b调节锌指蛋白13对骨肉瘤细胞增殖凋亡的影响

Mechanisms of microRNA-19b on osteosarcoma cell proliferation and apoptosis by Targeting Krueppel-like factor 13 Wang

目的观察微小RNA(miRNA,miR)-19b是否通过转录后上调癌基因低密度脂蛋白受Kruppel样因子13(KLF13)的表达而促进人骨肉瘤细胞增殖与凋亡.方法采用实时荧光定量聚合酶链反应(FQ-PCR)法检测人骨肉瘤细胞株和正常人成骨细胞中miR-19b与KLF13基因的表达;使用miR-19b抑制物(hsa-miR-19b inhibitor)抑制骨肉瘤细胞株中miR-19b的表达活性,应用荧光素酶活性实验和蛋白质印迹法(Western blot)检测miR-19b与靶基因KLF13表达调控的影响.并进一步采用四唑氮化合物(MTS)法和流式细胞仪检测MG-63细胞的增殖和凋亡变化.结果miR-19b及KLF13基因在骨肉瘤细胞株(MG-63、US-OS和Saos-2)及人成骨细胞(hFOB 1.19)相对表达分别为665.66±39.74、517.75±26.82、564.88±41.20和16.09±4.80,差异有统计学意义(均P<0.05).荧光素报告载体实验表明KLF13是miR-19b直接调控的靶基因.Western blot检测结果提示,导入反义寡核苷酸抑制miR-19b的表达后,KLF13的表达水平明显下降,对照组为0.783±0.053,实验组分别为0.424±0.021、0.265±0.016.进一步观察到此时骨肉瘤细胞MG-63的增殖能力显著抑制、凋亡显著增加,与阴性对照组比较差异有统计学意义(均P<0.01);而抑制KLF13的表达可以明显促进miR-19b介导的骨肉瘤细胞增殖和凋亡抑制.结论miR-19b可通过上调靶向癌基因KLF13的表达来促进骨肉瘤细胞的增殖和抑制骨肉瘤细胞的凋亡.

Objective To investigate the mechanisms of microRNA-19b on osteosarcoma cell proliferation and apoptosis by Targeting Krueppel-like factor 13(KLF13).Methods The expression of miR-19b and KLF13 was detected by quantitative Real-time polymerase chain reaction in three human osteosarcoma cell lines(MG-63,US-OS and Saos-2)and one normal human osteoblast cell line(hFOB 1.19).KLF13 as a potential target of miR-19b was predicted by sequence analysis.The viability and apoptosis effects of miR-19 inhibitor on MG-63 cell was assessed by Cell Proliferation Assay and flow cytometry.Luciferase assay and Western blotting were used to examine the regulatory effect.Results The relative expression of miR-19b and KLF13 in osteosarcoma cells(665.66±39.74,517.75±26.82,564.88±41.20)were significantly higher than that in normal human osteoblast cell(16.09±4.80)(both P<0.05).Luciferase reporter vector system confirmed that KLF13 was one of the target genes of miR一19b.The absorbance(4)of experiment groups and control group was 0.424±0.021,0.265±0.016 and 0.783±0.053 respectively by Western blotting.3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay and flow cytometry demonstrated that miR-19b inhibitor significantly inhibited proliferation and promoted apoptosis of MG-63 cell(both P<0.01).Moreover,the anti-proliferation and pro-apoptotic effect by miR-19b inhibitor could be consolidate d by KLF13 knock down.Conclusion Suppression the expression of miR-19b can effectively inhibit the proliferation and promote apoptosis of osteosarcoma cells.miR-19b may become a new target for the regulation of gene expression in osteosarcoma.