Zhongguo aizheng zazhi (Nov 2021)
Effect of CHKA gene silencing on biological behavior of glioma cell and its mechanism
Abstract
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 assessed through 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.
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