肺岩宁方介导Atg5对肺癌A549细胞自噬调控抗肺癌侵袭转移的实验研究

Experimental Study of Feiyan Ning Decoction Mediating Autophagy Regulation by Atg5 on Lung Cancer A549 Cells against Lung Cancer Invasion and Metastasis

目的探讨肺岩宁方(Feiyan Ning Decoction,FYN)通过介导Atg5(自噬相关靶向基因5,Autophagy-related gene 5)自噬相关基因调控人肺腺癌A549细胞自噬,及其对A549肺癌细胞增殖、迁移、侵袭等作用的影响。方法肺岩宁方干预培养肺癌A549细胞,通过运用CCK-8(Cell Counting kit-8)细胞增殖实验检测肺岩宁方对细胞增殖抑制率的影响,显微镜下观察肺岩宁方作用后的细胞形态学改变;采用划痕实验检测肺岩宁方及自噬抑制剂氯喹(Chloroquine diphosphate salt,CQ)对肺癌A549细胞迁移能力;运用Transwell细胞侵袭实验检测肺岩宁方及氯喹对肺癌A549细胞侵袭能力;运用Western Blot检测自噬相关蛋白Atg5、LC3表达情况;运用透射电子显微镜观察肺癌A549细胞在肺岩宁方干预下的自噬结构形态改变情况。结果CCK8检测试验结果:肺岩宁方能有效抑制A549细胞增值活力(P<0.01),其优化浓度为200μg mL-1;划痕实验结果:与对照组比,肺岩宁方干预A549细胞迁移能力减弱(P<0.05),A549细胞在FYN联合CQ的作用下迁移能力明显减弱(P<0.01);Transwell实验结果:与对照组比较,肺岩宁方抑制细胞侵袭转移能力(P<0.05),FYN联合CQ细胞侵袭力明显抑制(P<0.05);Western Blot实验结果:肺岩宁方下调自噬相关蛋白Atg5、LC3表达能力(P<0.01);透射电子显微镜观察结果:肺岩宁方抑制自噬结构的出现。结论肺岩宁方介导Atg5自噬相关基因,具有调控人肺腺癌A549细胞自噬能力,可抑制A549细胞增殖活力、迁移能力、侵袭转移水平。

Objective To investigate the mechanism of Feiyan Ning Decoction(FYN)regulating autophagy of lung cancer by mediating autophagy-related target gene Atg5,and its effects on proliferation,migration and invasion of lung cancer A549 cells.Methods Feiyan Ning Decoction was used to intervene lung cancer A549 cells,CCK-8 was used to detect the survival of lung cells treated with FYN.The morphological changes of A549 cells were observed under the microscope.The inhibitory effect of FYN and Chloroquine diphosphate salt(CQ)on the migration of A549 lung cancer cells were tested by scratch test.Transwell assay was used to detect the invasion ability of lung cancer A549 cells.Western Blot was used to detect the expression of autophagy related proteins Atg5 and LC3.The effects of Feiyan Ning on the autophagosome and autophagosome lysosomal structure of A549 lung cancer cells were observed by transmission electron microscopy(TEM).Results CCK8 showed that FYN effectively inhibited the proliferation activity of A549 cells(P<0.01),and its optimal concentration was 200μg·mL-1.Scratch showed that Compared with the control group,the ability of FYN to intervene A549 cells migration was reduced(P<0.05),and the group of drug combination was reduced obviously(P<0.01).Transwell assay result showed that FYN inhibited the ability of cells invasion and metastasis(P<0.05),and the group of drug combination inhibited the ability of cells invasion and metastasis(P<0.05).Western Blot showed that FYN down-regulated the expression of autophagy related proteins Atg5 and LC3(P<0.01).TEM showed that FYN interfered with the inhibition of autophagy.Conclusion Feiyan Ning Decoction mediates autophagy related genes Atg5,which can regulate the autophagy ability of lung cancer A549 cells,and inhibit the proliferation activity,migration ability,invasion and metastasis level of A549 cells.