Effect of siRNA-mediated gene silencing of transketolase on A549 lung cancer cells.
Effect of siRNA-mediated gene silencing of transketolase on A549 lung cancer cells.
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DOI：
10.3892/ol.2017.6916
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发表时间：
2017-11
期刊：
影响因子：
2.9
通讯作者：
Zhu H
中科院分区：
文献类型：
--
作者：
Lu H;Zhu H
本研究旨在探讨转酮醇酶(TKT)对A549肺癌细胞增殖、迁移及与其他代谢相关基因相互作用的影响。将3个TKT特异性的小干扰(Si)RNA导入A549细胞，筛选最适的转染浓度，用流式细胞仪和逆转录定量聚合酶链式反应(RT-qPCR)进行测序。用细胞计数试剂盒(CCK-8)检测细胞活力，用流式细胞仪检测细胞周期。细胞迁移通过划痕和Transwell小室实验确定。RT-qPCR检测葡萄糖-6-磷酸脱氢酶(G6PDH)、转醛酶(TAL)、山梨醇脱氢酶(SORD)、磷酸核糖焦磷酸合成酶1(PRPS1)和己糖激酶1(HK1)基因表达水平的变化。选择50nmol/L的siRNA-C进行后续实验。与阴性对照组相比，TKT-siRNA-C组细胞增殖受到明显抑制(CCK-8 24 h，0.2984±0.0371比0.0952±0.0063；P<；0.0001)，细胞周期停滞在G1/G0期(58±2.0%比70±2.5%；P=0.002)，细胞迁移能力降低[伤口大小，254.71±34.96比349.12±37.43µm(P=0.0001)；跨孔迁移，250±47.8/视野比150±49.0/视野(P<；0.0001)]。与阴性对照组相比，TKT-siRNA-C组G6PDH、TAL、SORD、PRPS1和HK1的mRNA表达水平下调。本研究表明，人工合成的TKT-siRNA能够抑制A549细胞的存活和迁移，这可能是由于细胞周期停滞和相关代谢酶下调所致。
The aim of the present study was to investigate the effects of transketolase (TKT) on cell proliferation, cell migration and interaction with other metabolism-associated genes in A549 lung cancer cells. A549 cells were transfected with three TKT-specific small interfering (si)RNAs, screened for the optimal transfection concentration, and sequenced with flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell viability was evaluated using Cell Counting Kit-8 (CCK-8), cell cycle was assessed by flow cytometric analysis. Cell migration was determined by scratch-wound and Transwell chamber assays. The changes in mRNA expression levels of glucose-6-phosphate dehydrogenase (G6PDH), transaldolase (TAL), sorbitol dehydrogenase (SORD), phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and hexokinase 1 (HK1) were detected by RT-qPCR. siRNA-C at 50 nmol/l was selected for the subsequent experiments. Compared with the negative control, cell proliferation of the TKT-siRNA-C group was inhibited dramatically (CCK-8 24 h, 0.2984±0.0371 vs. 0.0952±0.0063; P<0.0001), the cell cycle was arrested at the G1/G0 cell cycle phase (58±2.0% vs. 70±2.5%; P=0.002), and cell migration ability was decreased [wound size, 254.71±34.96 vs. 349.12±37.43 µm (P=0.0001); Transwell migration, 250±47.8/field vs. 150±49.0/field (P<0.0001)]. The mRNA expression levels of G6PDH, TAL, SORD, PRPS1 and HK1 were downregulated in the TKT-siRNA-C group compared with the negative control. The present study revealed that synthetic TKT-siRNA can inhibit A549 cell viability and migration, which may be due to arrest of the cell cycle and downregulation of relevant metabolic enzymes.
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