miR-3175/SIX5轴调节神经胶质瘤细胞的增殖和迁移

MiR-3175/SIX5 Axis Regulated Cell Proliferation and Migration of Glioma Cells

[目的]探讨miR-3175在胶质瘤细胞中的表达,以及对胶质瘤细胞增殖和迁移能力的影响及相关作用机制。[方法]采用实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)检测胶质瘤细胞与人正常胶质细胞中miR-3175的表达水平。向胶质瘤细胞株中转染miR-3175 mimics和inhibitors后,采用噻唑蓝(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT)法检测各组胶质瘤细胞增殖能力的变化,以Transwell实验对细胞迁移能力进行评估,流式细胞术检测细胞凋亡。使用miRTarBase和TargetScan预测软件对miR-3175的作用靶点进行预测,以双荧光素酶报告基因系统、免疫印迹法和Real-time qPCR分析miR-3175和sineoculis同源异型盒同源物5(sineoculis homeobox homologue 5,SIX5)的相互作用。通过生物信息学与免疫印迹法分析SIX5在胶质瘤中的表达;将pCDNA3.4-SIX5、pCDNA3.4-SIX5+miR-3175 mimics和miR-3175mimics分别转染到胶质瘤细胞,以MTT法、Transwell实验和流式细胞术检测miR-3175及其靶基因SIX5对胶质瘤细胞恶性生物学行为的影响。[结果] Real-time qPCR结果表明,胶质瘤细胞中miR-3175基因表达水平明显低于人正常胶质细胞组,呈低表达(P<0.01);细胞体外转染实验表明,上调miR-3175能够显著抑制胶质瘤细胞的增殖和迁移能力(P<0.05),能够通过将胶质瘤细胞的细胞周期阻滞于G0/G1期,诱导其凋亡(P<0.05,P<0.01);生物信息学软件预测结果显示SIX5可能是miR-3175的潜在靶基因,免疫印迹法、荧光素酶报告基因和Real-time qPCR结果进一步证明SIX5与miR-3175的表达呈负相关(P<0.01)。细胞体外转染实验结果发现,转染pCDNA3.4-SIX5后,胶质瘤细胞的增殖和迁移能力增加(P<0.05,P<0.01),凋亡率下降(P<0.05,P<0.01),而且可以削弱miR-3175对胶质瘤恶性生物学行为的抑制作用。[结论] miR-3175在胶质瘤细胞中显著低表达,并抑制胶质瘤的恶性生物学行为,可能的机制是抑制促癌基因SIX5的表达,从而降低胶质瘤的增殖和迁移能力,提示其具有成为胶质瘤诊疗新靶点的潜力。

[Objective] To investigate the expression of miR-3175 in glioma cells and its effect on the proliferation and migration of glioma cells and relevant mechanism. [Methods] The Real-time quantitative polymerase chain reaction(Real-time qPCR) was used to detect the expression of miR-3175 in glioma cells and human normal glial cells. After the miR-3175 mimics and inhibitors were transfected into A172 and U251 cells, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay was used to detect the proliferation of glioma cells in each group, the Transwell assay was used to detect the migration of cells, the flow cytometry was used to detect the apoptotic rate of cells. The target of miR-3175 was predicted by miRTarBase and TargetScan. Dual luciferase reporter gene system,Western blot and Real-time qPCR were used to detect the interaction between miR-3175 and sineoculis homeobox homologue 5(SIX5);bioinformatics and Western blot were used to analyze the expression of SIX5 in glioma. The pCDNA3.4-SIX5, pCDNA3.4-SIX5+miR-3175 mimics and miR-3175 mimics were transfected into glioma cells, MTT, Transwell and flow cytometry assays were performed to evaluate the effect of miR-3175 and its target gene SIX5 on the malignant biological behavior of glioma cells. [Results] The results of Realtime qPCR show that the expression level of miR-3175 is significantly lower in glioma cells than human normal glial cells(P<0.01). The results of vitro transfection experiments show that the up-regulation of miR-3175 could significantly inhibit the proliferation and migration of glioma cells(P<0.05), and induce apoptosis by arresting the cell cycle of glioma cells in the G0/G1 phase(P<0.05, P<0.01). The prediction results of bioinformatics show that SIX5 is probably a potential target gene of miR-3175. The Western blot analysis and luciferase reporter gene assay results further verify that the expression of SIX5 is negatively correlated with miR-3175(P<0.01). The results of vitro transfection experiments show that the proliferation and migration of glioma cells are significantly increased(P<0.05, P<0.01), while the apoptotic rate of glioma cells is significantly reduced after transfected pCDNA3.4-SIX5(P<0.05, P<0.01), overexpression of SIX5 can abrogate the inhibitory effect of miR-3175 on malignant biological behavior of glioma. [Conclusion] The expression level of miR-3175 is significantly downregulated in glioma, and miR-3175 inhibits the malignant biological behavior of glioma. The possible mechanism is to inhibit the expression of cancer promoting gene SIX5, so as to reduce the proliferation and migration ability of glioma, suggesting that it might have the potential to become a new target for glioma diagnosis and treatment.