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目的：通过特异性小干扰RNA(small interfering RNA, siRNA)，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(siRNA-CDK4)和阴性对照干扰片段(siRNA-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mRNA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mRNA 水平的变化。结果：将靶向CDK4 小干扰RNA(siRNA-CDK4)转染A549 细胞后，可明显抑制CDK4 的mRNA 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mRNA 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。

英文摘要:

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

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