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目的:通过特异性小干扰RNA(small interfering RNA,si RNA),使CDK4基因沉默,探讨该基因沉默对肺癌A549细胞增殖和代谢的影响及其可能的作用机制。方法:将靶向CDK4小干扰RNA(si RNA-CDK4)和阴性对照干扰片段(si RNA-control)成功转染A549细胞后,利用实时荧光定量PCR和蛋白质免疫印迹法分别检测CDK4在m RNA和蛋白水平的变化;细胞计数法、CCK-8法和软琼脂糖克隆形成实验检测A549增殖的变化和克隆形成能力;FCM法检测A549细胞的细胞周期;18F-FDG摄取实验、乳酸检测试剂盒及海马技术检测A549细胞中葡萄糖、乳酸的量及氧耗的变化;利用RT-PCR检测CDK4基因沉默后A549细胞中糖代谢相关酶m RNA水平的变化。结果:将靶向CDK4小干扰RNA(si RNA-CDK4)转染A549细胞后,可明显抑制CDK4的m RNA和蛋白表达(P<0.001,P<0.01)。CDK4蛋白抑制后,细胞增殖在48、72和96 h均明显降低(P值均<0.05),G1期细胞比例明显增多,S期细胞比例明显减少(P值均<0.05);18F-FDG摄取量下降(42.21±1.90)%(P<0.05),乳酸的生成量减少(29.39±5.35)%(P<0.05),而细胞的基础耗氧量增加(67.17±3.58)%(P<0.01);糖酵解相关酶PFKFB3、PKM2、LDHA在m RNA水平均明显减低(P<0.001,P<0.01,P<0.001)。结论:抑制CDK4表达可明显降低糖酵解水平,并增加耗氧量;同时可引起细胞周期阻滞,抑制肿瘤细胞增殖。其机制可能与CDK4直接或间接调节糖酵解相关酶的表达有关。
OBJECTIVE: To silence the CDK4 gene by siRNA (small interfering RNA, si RNA) and investigate its effect on proliferation and metabolism of lung cancer A549 cells and its possible mechanism. Methods: A549 cells were transfected with si RNA-CDK4 and si RNA-control siRNA targeting CDK4. The mRNA and protein expressions of CDK4 in m RNA and control group were detected by real-time fluorescence quantitative PCR and Western blot respectively. The changes of A549 cell proliferation and colony formation were measured by cell counting method, CCK-8 assay and soft agarose colony forming assay. The cell cycle of A549 cells was detected by FCM. The uptake of 18F-FDG, The changes of glucose and lactate amount and oxygen consumption in A549 cells were detected by hippocampus technique. The changes of glucose metabolism-related enzyme m RNA in A549 cells were detected by RT-PCR. Results: The siRNA targeting CDK4 siRNA transfected A549 cells significantly inhibited the expression of m RNA and protein of CDK4 (P <0.001, P <0.01). CDK4 protein inhibited cell proliferation at 48, 72 and 96 h (P <0.05), the proportion of cells in G1 phase increased significantly, and the proportion of S phase cells decreased significantly (all P <0.05). The uptake of 18F-FDG (P <0.05), the amount of lactic acid decreased (29.39 ± 5.35)% (P <0.05) and the basal oxygen consumption of cells increased (67.17 ± 3.58)% (P <0.01) (P <0.001, P <0.01, P <0.001). The level of m RNA in PFKFB3, PKM2 and LDHA were significantly decreased. Conclusion: Inhibition of CDK4 expression can significantly reduce the level of glycolysis and increase oxygen consumption; at the same time can cause cell cycle arrest and inhibit tumor cell proliferation. Its mechanism may be related to CDK4 directly or indirectly regulating the expression of glycolysis-related enzymes.