甲基莲心碱通过阻断ROCK通路抑制非小细胞肺癌细胞的迁移和侵袭

Effects of neferine on invasion and migration of non-small cell lung cancer H1299 cells via inhibiting ROCK pathway

目的验证甲基莲心碱(neferine,Nef)通过阻断ROCK通路对非小细胞肺癌H1299细胞迁移和侵袭的抑制作用。方法取H1299细胞体外培养,经不同浓度的Nef处理,CCK-8法检测H1299细胞活力确定实验组浓度。细胞划痕、Transwell小室实验检测H1299细胞的迁移和侵袭能力,酶联免疫吸附法检测肺癌细胞分泌的基质金属蛋白MMP-2、MMP-9的表达,实时荧光定量PCR和Western blot法检测H1299细胞ROCK1蛋白水平;应用AutoDock半柔性对接方法模拟了Nef与ROCK1之间的结合模式和亲合力。结果结论Nef作为一种潜在的抗癌天然化合物,通过下调与肿瘤细胞迁移和侵袭相关分子MMP-2、MMP-9和ROCK1的表达,进而发挥抑制非小细胞肺癌细胞迁移和侵袭的作用。

Aim To observe the inhibitory effect of neferine(Nef)on the migration and invasion of non-small cell lung cancer(NSCLC)H1299 cells by blocking ROCK pathway.Methods H1299 cells were taken for in vitro culture,and treated with different concentrations of Nef.H1299 cell viability was measured by CCK-8 method to determine the dose of the experimental group.The migration and invasion abilities of H1299 cells were detected by cell scratch test and Transwell chamber test.The expression of matrix metalloproteinases MMP-2 and MMP-9 secreted from lung cancer cells was detected by enzyme linked immunosorbent assay(ELISA).The protein level of ROCK1 in H1299 cells was tested by real-time fluorescent quantitative PCR and Western blot;the binding mode and affinity between Nef and ROCK1 were stimulated by AutoDock semi flexible docking method.Results The doses of Nef in the experimental group were determined as 4,6 and 10μmol·L-1.These three concentrations of Nef could inhibit the migration and invasion of H1299 lung cancer cells to a certain degree in a dose-dependent manner.At the same time,Nef reduced the expression of MMP-2,MMP-9 and ROCK1 proteins related to the migration and invasion of the cancer cells.In addition,the affinity of Nef to ROCK1 was significantly higher than that of fasudil,an inhibitor of ROCK,and the binding force was stronger to A-chain of ROCK1.Conclusions As a potential natural anticancer compound,Nef can inhibit the migration and invasion of NSCLC by reducing the expression of MMP-2,MMP-9 and ROCK1 proteins related to the migration and invasion of the cancer cells.