CHKA基因沉默对胶质瘤细胞生物学行为的影响及其机制

背景与目的：胶质瘤是颅内常见的原发性恶性肿瘤之一，但发病机制不明，胆碱激酶A（choline kinase A，CHKA）与胶质瘤的恶性程度呈正相关，但具体作用途径尚不清楚。探讨下调CHKA基因的表达对胶质瘤细胞系U87和U251增殖、凋亡和迁移的影响及其作用机制。方法：使用慢病毒感染胶质瘤细胞系U87和U251，同时设置阴性对照组（shNC组）和空白对照组（control组）。为研究磷脂酰肌醇3-激酶（phosphoinositide 3-kinase，PI3K）/蛋白激酶B（protein kinase B，AKT）信号转导通路与CHKA的相互作用，另设DMSO组和LY294002组。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）检测CHKA基因mRNA的表达水平，采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）法检测细胞增殖能力，采用流式细胞术检测细胞周期和凋亡，采用transwell实验检测细胞侵袭能力，采用划痕实验检测细胞迁移能力，采用蛋白质印迹法（Western blot）检测胶质瘤细胞中CHKA、PI3K、p-PI3K、AKT和p-AKT蛋白的水平。结果：Western blot结果显示，与control组和shNC组相比，shCHKA组胶质瘤细胞中p-PI3K、p-AKT和CHKA蛋白水平同步降低（P＜0.05），PI3K、AKT蛋白水平无明显改变。在使用通路抑制剂后CHKA的表达量在control组和shNC组之间差异无统计学意义（P＞0.05）。与此同时，shCHKA组胶质瘤细胞的侵袭、迁移能力均弱于control组和shNC组（P＜0.05），细胞凋亡率明显增加，细胞周期主要停滞于G ₂ 期，细胞增殖明显降低（P＜0.05）。结论：CHKA基因可能是通过调控PI3K/AKT信号转导通路，从而影响胶质瘤细胞增殖、凋亡、迁移和侵袭等生物学行为，且CHKA对PI3K/AKT信号转导通路的调控是单向的，PI3K/AKT信号转导通路中蛋白水平的降低对CHKA的表达无反馈作用。

Effect of CHKA gene silencing on biological behavior of glioma cell and its mechanism

Background and purpose: Glioma is one of the common intracranial malignant tumors, but the specific mechanism was unclear. The expression level of choline kinase A (CHKA) is positively correlated with the malignant grade of glioma. However, the specific mechanisms by which CHKA acts are also unclear. The aim of the present study was to evaluate the effects of CHKA gene downregulation on proliferation, migration, invasion, apoptosis of U87 and U251 cells and elucidate the possible underlying mechanism. Methods: A lentiviral vector was utilized to stably knockdown CHKA gene in U87 and U251 cell lines, and the shNC group and control group were set up. To determine the interaction between CHKA and phosphoinositide 3-kinase (PI3K)/protein kinase (AKT) signal pathways, DMSO group and LY294002 group were established. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was used to measure CHKA gene mRNA expression levels. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay. Cell cycle and apoptosis were analyzed by flow cytometer. Cell invasion ability was assessedthrough transwell assay. The ability of cell migration was detected using wound scratch assay. CHKA, PI3K, p-PI3K, AKT and p-AKT expressions at the protein level were evaluated using Western blot. Results: Western blot results showed the expressions of p-PI3K, p-AKT and CHKA in shCHKA group were further decreased (P＜0.05), while expressions of PI3K and AKT did not change significantly (P＞0.05) compared with the Control group and shNC group. There was no difference in expression of CHKA between the Control group and shNC group after the administration of PI3K/AKT inhibitor (LY294002) (P＜0.05). Concurrently, compared with the Control group and shNC group, cells in the shCHKA group exhibited significantly lower cell viability and invasive ability, and had a significantly higher apoptotic rate. glioma cells mainly remained arrested in the G2 phase of the cell cycle (P＜0.05). Conclusion: The results demonstrated that CHKA gene may function by altering PI3K/AKT signal transduction pathway, knockdown of CHKA in U251 and U87 cells to inhibit cell proliferation, migration and invasion, while promoting apoptosis. These results reveal that CHKA directly and positively regulates PI3K/AKT signaling pathways without feedback inhibition of CHKA expression by PI3K/AKT.