| 申梦琴,赵小平,赵 丽,黄 钢,刘建军.沉默TIGAR基因对非小细胞肺癌细胞增殖和代谢的影响[J].现代生物医学进展英文版,2018,(7):1201-1206. |
| 沉默TIGAR基因对非小细胞肺癌细胞增殖和代谢的影响 |
| Effects of TIGAR Knockdown on the Proliferation and Metabolism of Human Non-small Cell Lung Cancer |
| Received:December 08, 2017 Revised:December 28, 2017 |
| DOI：10.13241/j.cnki.pmb.2018.07.001 |
| 中文关键词: 非小细胞肺癌 TIGAR RNA干扰 细胞增殖 代谢流量 |
| 英文关键词: Non-small cell lung cancer TIGAR RNA interference Cell Proliferation Metabolism flux |
| 基金项目:国家自然科学基金项目（81471687） |
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| 中文摘要: |
| 摘要 目的：通过沉默TIGAR(Tp53 induced glycolysis and apoptosis regulator)基因，探讨其对非小细胞肺癌细胞增殖及代谢的影响及可能的机制。方法：用靶向TIGAR的siRNA沉默TIGAR，采用实时荧光定量PCR(RT-qPCR)及蛋白印迹(WB)分别检测TIGAR mRNA和蛋白表达水平，将敲除效率较好的敲除序列包装进重组慢病毒感染细胞构建稳定敲减TIGAR的细胞。通过CCK-8法、软琼脂克隆形成、FCM法分别检测细胞的增殖速率、克隆形成能力和细胞周期分布；蛋白印迹法检测TIGAR沉默后周期相关蛋白CDK4和p27的表达情况。RT-qPCR检测TIGAR对糖代谢相关酶表达的影响；18F-FDG、1-14C、6-14C摄取实验分别检测相应的代谢流量，乳酸试剂盒和ROS试剂盒分布检测细胞乳酸生成和ROS水平。结果：转染siTIGAR或感染shTIGAR病毒后细胞TIGAR表达水平明显下降(P值均＜0.005)。成功构建了两株稳定敲减TIGAR的非小细胞肺癌细胞株，shTIGAR组细胞增殖速率和克隆形成能力明显下降，细胞周期阻滞在G0/G1期，P27蛋白明显上调，CDK4蛋白明显下调。沉默TIGAR促进细胞糖酵解速率和乳酸生成，抑制磷酸戊糖途径和氧化磷酸化，细胞ROS生成增加尤其是低氧情况下(P值＜0.05)。结论：下调TIGAR表达的非小细胞肺癌细胞增殖能力下降，其机制可能与调控CDK4和P27表达及代谢流量再分布相关。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effect of TIGAR on the proliferation and metabolism flux in non-small cell lung cancer and explore its possible mechanisms. Methods: A549 cells were transfected with two pair of siTIGAR or siNC (as the control). The effi- ciency of TIGAR silencing was detected by real-time fluorescence quantitative PCR and Western blotting, respectively. The effective shTIGAR was constructed into a recombinant lentiviral vector, and then infected cells. The proliferation ability, colony formation, cell cycle was detected by CCK-8 assay, soft agar assay and FCM assay, respectively. Western blotting determined the effect of TIGAR on the protein expression of P27 and CDK4. The mRNA levels of several glycolysis enzyme was detected by RT-qPCR when knockdown TIGAR. 18F-FDG intake, 1-14C intake, 6-14C intake assay indicated relevant metabolism flux. Lactate and ROS was quantified by kits. Results: The expression of TIGAR mRNA and protein were strongly decreased after transfection with siTIGAR (both P value＜0.005). The NSCLC cells stably knockdown TIGAR were successfully constructed. The depressed proliferation and cloning ability, arrested in G0/G1 phase were found in stably knockdowned-TIGAR cells. The protein of P27 was upregulated while CDK4 was downregulated by TIGAR knockdown. TIGAR gene silencing significantly promoted the glycolysis and lactate production and inhibited the flux of pentose phosphate pathway and oxidative phosphorylation. Inhibition of TIGAR induced the ROS Production, especially in the hypoxia status. Conclusion: TIGAR silencing may inhibit the proliferation via regulating the P27 and CDK4 expressions and the redistribution of metabolism flux. |
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