LncRNA ZFAS1对NCI-H460细胞增殖、侵袭及化疗敏感性的影响

Effect of LncRNA ZFAS1 on the proliferation,invasion and chemotherapy sensitivity of NCI-H460 cells

目的研究LncRNA ZFAS1调控肺癌NCI-H460细胞增殖、侵袭和化疗敏感性的作用机制。方法收集肺癌组织、癌旁正常组织、NCI-H460细胞和BEAS-2B细胞,通过实时荧光定量逆转录聚合酶链反应(RT-qPCR)分析LncRNA ZFAS1、miR-589-5p的表达量。miRcode软件预测LncRNA ZFAS1和miR-589-5p的结合位点,进一步通过双荧光素酶报告基因确定两者的相关性。以细胞计数法-8(CCK-8)实验检测NCI-H460细胞增殖活力,以Transwell实验检测细胞侵袭数目,以流式细胞仪检测细胞凋亡率,以蛋白质印迹法检测丝裂原活化蛋白激酶/细胞外信号调节激酶(MAPK/ERK)通路蛋白表达。结果NCI-H460细胞(3.02±0.98)和肺癌组织(2.34±0.67)中LncRNA ZFAS1表达量明显升高(P<0.01),NCI-H460细胞(0.34±0.03)和肺癌组织(0.23±0.01)中miR-589-5p的表达量明显降低(P<0.01)。ZFAS1 siRNA和siRNA NC组细胞增殖活力分别为(37.52±2.58)%,(62.38±3.55)%,细胞侵袭数目分别为(32.50±4.52),(83.50±5.86)个,差异有统计学意义(P<0.05,P<0.01)。siRNA NC+8μg·mL^(-1)顺铂组、ZFAS1 siRNA+8μg·mL^(-1)顺铂组细胞凋亡率分别为(8.73±2.17)%,(20.06±1.85)%,差异有统计学意义(P<0.01)。LncRNA ZFAS1与miR-589-5p具有靶向结合位点。下调miR-589-5p激活了MAPK/ERK通路。inhibitor NC组、miR-589-5p inhibitor组、miR-589-5p inhibitor+U0126组细胞增殖活力分别为(63.18±3.39)%,(82.35±4.58)%,(60.27±3.93)%,细胞侵袭数目分别为(75.50±6.85),(135.50±5.83),(90.50±6.84)个,差异均有统计学意义(均P<0.01)。inhibitor NC+8μg·mL^(-1)顺铂组、miR-589-5p inhibitor+8μg·mL^(-1)顺铂组、miR-589-5p inhibitor+U0126+8μg·mL^(-1)顺铂组细胞增殖活力分别为(38.25±2.78)%,(75.45±4.28)%,(42.85±5.87)%,细胞凋亡率分别为(12.59±4.15)%,(5.26±2.48)%,(11.06±2.45)%,差异均有统计学意义(均P<0.01)。结论LncRNA ZFAS1在肺癌细胞中表达上调且通过miR-589-5p激活MAPK/ERK通路调控NCI-H460细胞增殖、侵袭及化疗敏感性。

Objective To investigate the mechanism of lncRNA ZFAS1 regulating the proliferation,invasion and chemosensitivity of lung cancer NCI-H460 cells.Methods Lung cancer tissues,adjacent normal tissues,NCI-H460 cells and BEAS-2B cells were collected,and the expression levels of LncRNA ZFAS1 and miR-589-5p were analyzed by reverse transcription quantitative polymerase chain reaction(RT-qPCR).The miRcode software predicted the binding sites of LncRNA ZFAS1 and miR-589-5p,and further determined the correlation between the two by double luciferase reporter genes.Cell counting kit-8(CCK-8)assay was used to detect the proliferation activity of NCI-H460 cells;Transwell assay was used to detect the number of cells invasion;flow cytometry was used to detect the cell apoptosis rate;Western blot was used to detect the protein expression of mitogen activated protein kinases/extracellular signal-regulated kinase(MAPK/ERK)pathway.Results LncRNA ZFAS1 expression was significantly increased in NCI-H460 cells(3.02±0.98)and lung cancer tissues(2.34±0.67,P<0.01).The expression of miR-589-5p was significantly decreased in NCI-H460 cells(0.34±0.03)and lung cancer tissues(0.23±0.01,P<0.01).The proliferation activity of ZFAS1 siRNA and siRNA NC groups were(37.52±2.58)%,(62.38±3.55)%;the number of cell invasion were 32.50±4.52 and 83.50±5.86;the differences were statistically significant(P<0.05,P<0.01).The apoptosis rates of siRNA NC+8μg·mL^(-1) cisplatin group and ZFAS1 siRNA+8μg·mL^(-1) cisplatin group were(8.73±2.17)%and(20.06±1.85)%;the difference was statistically significant(P<0.01).LncRNA ZFAS1 has a targeted binding site with miR-589-5p.Downregulation of miR-589-5p activated the MAPK/ERK pathway.Proliferation activity of cells in inhibitor NC group,miR-589-5p inhibitor group,miR-589-5p inhibitor+U0126 group were(63.18±3.39)%,(82.35±4.58)%,(60.27±3.93)%;the number of cell invasion were 75.50±6.85,135.50±5.83,90.50±6.84;the differences were statistically significant(P<0.01).Cell proliferation activity in inhibitor NC+8μg·mL^(-1) cisplatin group,miR-589-5p inhibitor+8μg·mL^(-1) cisplatin group,miR-589-5p inhibitor+U0126+8μg·mL^(-1) cisplatin group were(38.25±2.78)%,(75.45±4.28)%,(42.85±5.87)%;apoptosis rates were(12.59±4.15)%,(5.26±2.48)%,(11.06±2.45)%;the differences were statistically significant(all P<0.01).Conclusion LncRNA ZFAS1 was up-regulated in lung cancer cells and regulated the proliferation,invasion and chemotherapy sensitivity of NCI-H460 cells through miR-589-5p activation of MAPK/ERK pathway.