金粉蕨素通过circ_0136666/miR-370轴对肺癌A549细胞糖酵解、增殖、迁移和侵袭的影响

Effects of onychin on the glycolysis,proliferation,migration and invasion of lung cancer A549 cells via circ_0136666/miR-370 axis

目的探讨金粉蕨素(ONY)对肺癌A549细胞糖酵解、增殖、迁移和侵袭的影响及其作用机制。方法体外培养A549细胞,用不同浓度(5、10、20μg·mL^(-1))ONY作用24 h,转染circ_0136666的小干扰RNA至A549细胞,或将20μg·mL^(-1)ONY作用于转染circ_0136666过表达载体的A549细胞。酶联免疫吸附法检测细胞中的葡萄糖水平及细胞培养上清中的乳酸含量,CCK-8检测细胞增殖,Transwell检测细胞迁移和侵袭,Western blotting检测Ki-67、MMP-2和MMP-9蛋白表达,RT-qPCR检测circ_0136666和miR-370表达,双荧光素酶报告基因实验验证A549细胞中circ_0136666和miR-370的调控关系。结果经5、10、20μg·mL^(-1)ONY干预后,A549细胞葡萄糖消耗量和乳酸产生量、细胞迁移数和侵袭数、Ki-67、MMP-2和MMP-9蛋白表达水平及circ_0136666表达水平明显降低(P<0.05),而细胞增殖抑制率、miR-370表达水平明显升高(P<0.05),且具有剂量依赖性。circ_0136666可靶向负调控miR-370的表达。沉默circ_0136666后,A549细胞葡萄糖消耗量和乳酸产生量、细胞迁移和侵袭数、Ki-67、MMP-2和MMP-9蛋白表达水平明显降低(P<0.05),而细胞增殖抑制率显著升高(P<0.05)。过表达circ_0136666逆转了ONY对A549细胞糖酵解、增殖、迁移和侵袭的抑制作用。结论ONY可抑制肺癌A549细胞的糖酵解、增殖、迁移和侵袭,其作用机制与调控circ_0136666/miR-370通路有关。

Objective To investigate the effect of onychin(ONY)on glycolysis,proliferation,migration and invasion of lung cancer A549 cells and its mechanism.Methods A549 cells were cultured in vitro.After treated with different concentrations of ONY(5,10,20μg·mL^(-1))for 24 h,A549 cells were transfected with circ_0136666 small interfering RNA.The A549 cells transfected with circ_0136666 over-expression vector were treated with 20μg·mL^(-1) ONY.The enzyme-linked immunosorbent assay was used to detect the glucose level in cells and the lactic acid content in the cell culture supernatant.CCK-8 was used to detect the cell proliferation,and Transwell was used to detect the cell migration and invasion.The protein expression levels of Ki-67,MMP-2 and MMP-9 were detected by Western blotting,and the expression levels of circ_0136666 and miR-370 were detected by RT-qPCR.The regulatory relationship between circ_0136666 and miR-370 in A549 cells was verified by the dual luciferase reporter gene assay.Results After A549 cells were interfered with 5,10,20μg·mL^(-1) ONY,the glucose consumption and lactic acid production of the cells,were significantly reduced,as well as the number of migration and invasion cells,the protein expression levels of Ki-67,MMP-2 and MMP-9,and the expression level of circ_0136666(P<0.05),while the cell inhibition rate and the expression level of miR-370 were significantly increased(P<0.05),and all were dose-dependent.The circ_0136666 negatively regulated the expression of miR-370.After silencing circ_0136666,the glucose consumption and lactic acid production of the cells,the number of migration and invasion cells,and the protein expression levels of Ki-67,MMP-2 and MMP-9 all were reduced(P<0.05),while the cell inhibition rate was increased(P<0.05).Overexpression of circ_0136666 partially restored the inhibitory effect of ONY on glycolysis,proliferation,migration and invasion of A549 cells.Conclusion ONY could inhibit the glycolysis,proliferation,migra-tion and invasion of lung cancer cell A549,and its mechanism of action may be related to the regulation of circ_0136666/miR-370 pathway.