CDK4基因沉默对非小细胞肺癌A549 增殖和代谢的影响

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(P0.001,P0.01)。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)%(P0.05),乳酸的生成量减少(29.39±5.35)%(P0.05),而细胞的基础耗氧量增加(67.17±3.58)%(P0.01);糖酵解相关酶PFKFB3、PKM2、LDHA在m RNA水平均明显减低(P0.001,P0.01,P0.001)。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。

The Effects of CDK4 Gene Silencing on the Proliferation and Metabolismin 549 Cells

Objective:To investigate the effect of CDK4 gene silencing on proliferation and metabolismof human lung cancer cellline A549, and to explore its possible mechanisms.Methods:The specific small interfering RNA targeting CDK4 gene and negativecontrol were designed and synthesized and then were transfected into the human lung cancer cell line A549. The efficiency of CDK4gene silencing in mRNA and protein levels was detected by real-time fluorescent quantitative PCR and Western blot, respectively. Thechanges in proliferation, colony formation and cell cycle were observed by cell count, CCK-8 assay, colony formation assay and flowcytometry, respectively. The changes in glucose metabolism and oxygen consumption were detected by 18 F-FDG uptake assay, lactatedetection assay and Seahorse XF24 analyzer assay. Meanwhile, the expression levels of metabolism related factors were detected byreal-time fluorescent quantitative PCR.Results:After the siRNA-CDK4 was successfully transfected into A549 cells, the expressionlevels of CDK4 mRNA and protein were significantly lower than that of the negative control (P < 0.001, P < 0.01). Compared with thenegative control (siRNA-NC) group, the group transfected with siRNA-CDK4 showed that the cell proliferation in 48, 72 and 96h wasdecreased (P < 0.05), the cell cycle was arrested in G1 stage and the percentage of S stage was decreased (both P < 0.05). The 18F-FDGuptake was decreased (42.21± 1.90)% (P < 0.05), and lactate production was reduced (29.39± 5.35)% (P < 0.05), but basic oxygenconsumption was increased (67.17± 3.58)% (P < 0.01). Besides, the expression level of the glycolysis related factors mRNA wasdeclined, such as PFKFB3, PKM2, LDHA (P < 0.001, P < 0.01, P <0.01).Conclusion:CDK4 gene silencing can significantly inhibitglycolysis level and increase oxygen consumption, arrest the cell cycle in G1 stage and inhibit the cell proliferation, and its mechanismmay be related to the regulation of expression of PFKFB3, PKM2 and LDHA.