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目的:探讨微小RNA(microRNA,miRNA)let-7a对尤文氏肉瘤A673和SK-ES-1细胞生物学行为的影响及其可能的作用机制。方法:将成熟的let-7a片段(has-miR-let-7a mimic)转染至A673和SK-ES-1细胞中,实验分为转染组(转染has-miR-let-7a mimic)、对照组(转染has-miR-let-7a mimic-control)和未转染组(仅加入脂质体)。Has-miR-let-7a mimic转染后,应用实时荧光定量-PCR法检测细胞中let-7a的表达,CCK-8法检测各组细胞的增殖活性,FCM法检测细胞的周期分布和凋亡变化,Transwell小室检测细胞的迁移和侵袭能力,蛋白质印迹法检测细胞中细胞周期依赖性激酶6(cyclin dependent kinase 6,CDK6)、Rb和磷酸化-Rb(phospho-Rb,p-Rb)蛋白的表达。结果:与未转染组和对照组比较,转染has-miR-let-7a mimic后的A673和SK-ES-1细胞中let-7a的表达水平明显上调(P<0.01),细胞增殖受到抑制(P<0.01),G0/G1期细胞所占比例上升(P<0.01),细胞的早期凋亡率明显升高(P<0.01),细胞的迁移和侵袭能力明显下降(P<0.01),细胞中CDK6和p-Rb蛋白的表达水平明显下降(P<0.01),而Rb蛋白的表达水平无明显变化。结论:Let-7a可抑制尤文氏肉瘤A673和SK-ES-1细胞的增殖、迁移和侵袭,并促进其凋亡,这一作用可能部分依赖于let-7a靶向抑制CDK6的表达。
Objective: To investigate the effect of let-7a microRNA (miRNA) on the biological behavior of Ewing’s sarcoma A673 and SK-ES-1 cells and its possible mechanism. Methods: The mature let-7a fragment (has-miR-let-7a mimic) was transfected into A673 and SK-ES-1 cells. , Control group (transfected with has-miR-let-7a mimic-control) and untransfected group (liposome only). After transfected with Has-miR-let-7a mimic, the expression of let-7a in the cells was detected by real-time fluorescence quantitative PCR. The proliferation activity of the cells was detected by CCK-8 assay. The cell cycle distribution and apoptosis Transwell chamber was used to detect the migration and invasion ability of cells. Western blotting was used to detect the expression of cyclin dependent kinase 6 (CDK6), Rb and phospho-Rb (p-Rb) expression. Results: The expression of let-7a in A673 and SK-ES-1 cells transfected with has-miR-let-7a mimic was significantly up-regulated (P <0.01) compared with untransfected and control groups, (P <0.01). The proportion of cells in G0 / G1 phase increased (P <0.01), the early apoptosis rate increased significantly (P <0.01) and the migration and invasion ability of cells decreased significantly (P <0.01) , While the expression of CDK6 and p-Rb in the cells was significantly decreased (P <0.01), while the expression level of Rb protein did not change significantly. Conclusion: Let-7a inhibits the proliferation, migration, invasion and apoptosis of Ewing’s sarcoma A673 and SK-ES-1 cells, and this effect may be partly dependent on the let-7a target inhibition of CDK6 expression.