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目的:研究人骨肉瘤细胞系(Saos-2,MG-63)中microRNA-150(miR-150)表达水平及其对细胞增殖的作用,并探讨其分子生物学作用机制。方法:采用相对实时定量PCR检测人骨肉瘤细胞系Saos-2,MG-63和正常成骨细胞系NHOst中miR-150表达差异。采用慢病毒转染过表达miR-150,实时定量PCR检测过表达组miR-150水平。细胞增殖实验采用MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]法。Western印迹检测Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)及内参β-actin蛋白水平。双荧光素酶反应检测miR-150与RUNX2 3’-UTR结合对上游基因表达抑制率。结果:MiR-150在Saos-2和MG-63细胞系中表达低于正常成骨细胞系NHOst,分别为0.23±0.02,0.32±0.03和1.00±0.02,差异具有统计学意义(P<0.01)。Saos-2细胞系空载体对照组和miR-150过表达组分别为1.00±0.08和4.17±0.19,差异具有统计学意义(P<0.01),MG-63细胞系空载体对照组和miR-150过表达组分别为1.00±0.07和3.43±0.10,差异具有统计学意义(P<0.01)。过表达miR-150后,Saos-2和MG-63细胞增殖率明显下降(P<0.01),RUNX2蛋白量明显降低(P<0.01)。Saos-2细胞系中,miR-150结合RUNX2 3’-UTR区抑制69%上游荧光素酶活性(P<0.05);MG-63细胞系中,miR-150抑制59%上游荧光素酶活性(P<0.05)。在过表达miR-150的Saos-2和MG-63细胞中,补充外源性RUNX2蛋白,可恢复骨肉瘤细胞的增殖水平(P<0.01)。结论:MiR-150具有抑制骨肉瘤细胞增殖的作用,直接结合RUNX2 3’-UTR抑制转录因子RUNX2合成是其重要的作用机制。
Objective: To investigate the expression of miR-150 in human osteosarcoma cell line (Saos-2, MG-63) and its effect on cell proliferation and to explore its molecular biological mechanism. Methods: The relative real-time PCR was used to detect the expression of miR-150 in human osteosarcoma cell lines Saos-2, MG-63 and normal osteoblastic cell line NHOst. MiR-150 was overexpressed by lentivirus and miR-150 level was detected by real-time quantitative PCR. Cell proliferation assay was performed using MTS [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium] Western blotting was used to detect the expression of Runt-related transcription factor 2 (RUNX2) and β-actin protein. Dual-luciferase assay was used to detect the inhibition of upstream gene expression by combining miR-150 with RUNX2 3’-UTR. Results: The expression of MiR-150 in Saos-2 and MG-63 cell lines was lower than that in normal osteoblast cell line NHOst (0.23 ± 0.02,0.32 ± 0.03 and 1.00 ± 0.02, respectively) (P <0.01) . The expression of miR-150 in empty vector control group and miR-150 overexpression group was significantly higher than that in miR-150 overexpression group (P <0.01) Overexpression group were 1.00 ± 0.07 and 3.43 ± 0.10, the difference was statistically significant (P <0.01). After overexpression of miR-150, the proliferation rate of Saos-2 and MG-63 cells was significantly decreased (P <0.01), and the amount of RUNX2 protein was significantly decreased (P <0.01). In the Saos-2 cell line, miR-150 bound 69% of the upstream luciferase activity (P <0.05) in the RUNX2 3’-UTR region and 59% of the upstream luciferase activity in the MG-63 cell line P <0.05). In exogenous miR-150-overexpressing Saos-2 and MG-63 cells, exogenous RUNX2 protein could restore the proliferation of osteosarcoma cells (P <0.01). Conclusion: MiR-150 can inhibit the proliferation of osteosarcoma cells. Direct binding of RUNX2 3’-UTR inhibits transcription of RUNX2 is an important mechanism of action.