关键词: 尤文肉瘤, RNA干扰, 细胞周期蛋白依赖激酶6

Abstract:

Background and purpose: Cyclin-dependent kinase 6 (CDK6) is a kind of serine/threonine protein kinase, which has been reported to be over-expressed in the Ewing’s sarcoma cells. However, the biological effects and relative mechanisms of CDK6 on Ewing’s sarcoma cells are really less known. This study aimed to investigate the effects of CDK6 gene on the biological behaviors of Ewing’s carcinoma cells A673 and SK-ES-1, we knocked down CDK6 using a small interfering RNA targeting CDK6. Methods: The A673 and SK-ES-1 cells were transfected with a chemical synthesized small interfering RNA which targets CDK6; The real time-PCR and Western blot assays were performed to detected the mRNA and protein expression levels of CDK6 and its downstream genes Rb and P-Rb in both cells transfected with the siRNAs; The CCK-8, flow cytometry (FCM), migration and invasion assays were performed to indentify the proliferation, cell cycle distribution, apoptosis, migration and invasion abilities after the transfection with si-CDK6, respectively. Results: The real time-PCR analysis showed the expression of CDK6 was down-regulated in the A673 and SK-ES-1 cells transfected with si-CDK6 (P<0.01); Suppression of CDK6 by si-CDK6 inhibited the cell proliferation, induced cell apoptosis and G₀/G₁-Phase cell cycle arrest (P<0.01); The cells of A673 migrating through the membrane in untreated group, si-con group and si-CDK6 group were 516.00±58.59, 534.00±124.77 and 192.33±68.92, respectively. The numbers of SK-ES-1 cells were 371.33±29.67, 363.33±60.28 and 200.00±20.00, respectively. The results showed significant differences in statistics (P<0.01); In the invasion assay, the cells of A673 passed through the Matrigel coated membrane in untreated group, si-con group and si-CDK6 group were 251.00±42.93, 238.67±78.62 and 94.67±23.03, respectively, and the number of SK-ES-1 cells were 310.00±35.36, 302.33±41.31 and 105.00±54.08, respectively. The results showed down-regulation of CDK6 could suppress the cells migration and invasion abilities (P<0.01); The Western blot analysis showed the protein levels of CDK6 and its downstream gene phospho-retinoblastoma gene (P-Rb) were down-regulated, while there were no changes in the expression of Rb after transfection.Conclusion: CDK6 siRNA specifically and efficiently blocks the constitutively activated CDK6 in human Ewing’s sarcoma cells, resulting in inhibition in cell proliferation, migration, invasion and induction of cell apoptosis.

Key words: Ewing’s sarcoma, RNA interference, CDK6