miRNA-145抑制肺癌A549细胞增殖活性的机制

Mechanism of miRNA-145 inhibits cell proliferation of A549 cells

目的:研究miRNA-145(miR-145)抑制肺癌A549细胞增殖活性的机制。方法:应用生物信息学方法预测Sp1基因3'-UTR区与miR-145的结合位点并通过荧光素酶报告基因法进行验证;转染miRNA-145拟似物和阻遏物到A549细胞,实时荧光定量PCR法检测细胞内miR-145含量,蛋白质印迹法检测细胞中Sp1蛋白含量;同时采用MTT法检测转染后24,48和72 h A549细胞的增殖活性。结果:荧光素酶报告基因系统验证结果表明,成功预测了miR-145与人Sp1基因3'-UTR区的结合位点。与单独转染组比较,miR-145拟似物和miR-145阻遏物与野生型荧光素酶报告基因共转染可以明显抑制或增强荧光素酶活性(P<0.05)。转染miR-145拟似物和阻遏物能明显抑制或者促进A549细胞内Sp1蛋白的表达(P均<0.05)。实时荧光定量检测结果表明,转染后48 h,与转染对照组比较,拟似物组细胞内miR-145含量明显上升,而阻遏组细胞内miR-145含量明显下降(P均<0.05),阴性对照转染组和转染对照组miR-145相对含量间的差异无统计学意义(P>0.05)。细胞活性检测结果表明,转染72 h后,拟似物组细胞增殖活性明显降低,而阻遏组细胞活性明显增高(P均<0.05)。结论:miR-145通过抑制Sp1基因表达,明显抑制A549细胞的增殖活性。

Objective: To investigate the mechanism underlying how miR-145 inhibits cell proliferation in A549 cells. Methods: The putative miR-145 binding sites in the 3’-UTR region of Sp1 gene were predicated by bioinformatics and was predicted by a luciferase method. miR-145 levels were measured by q PCR and Sp1 protein levels were measured by Western blotting after transiently transfected with either miR-145 mimics or inhibitors into A549 cells. MTT assays were employed to evaluate cell proliferation in A549 cells simultaneously at 24,48 and 72 h after transfection. Results: Through report of luciferase analysis,the study successfully predicted and verified the putative miR-145 binding site in the 3’-UTR of human Sp1 gene. The luciferase activity of the wild type is reported that it could be significantly reduced and induced48 h post-transfection of miR-145 mimics and inhibitors respectively and it had significant difference between the above two groups( P < 0. 05). Compared to the control,transfection of miR-145 mimics or miR-145-inhibitors reduced or induced Sp1 protein levels with the significant difference( P < 0. 05). Data from real-time q PCR indicated that transfection of miR-145-mimics and inhibitors induced and reduced miR-145 levels respectively compared to the negative control with a significant difference( P < 0. 05). There was no significant difference between the negative control transfection group and transfection control group( P >0. 05). Finally,transfection of miR-145-mimics and miR-145-inhibitor could significantly reduce and induce cell proliferation of A549 cells. Seventy-two hours post transfection,there had significant difference between miR-145-mimics transfection group and the negative control transfection group( P < 0. 05). Conclusion: miR-145 inhibited cell proliferation of A549 cells through reducing Sp1 gene expression.