长链非编码RNA结直肠肿瘤差异表达基因靶向微小RNA-384对肺癌SPC-A1细胞侵袭和迁移的影响

Effect of long non-coding RNA colorectal neoplasia differentially expressed targeting microRNA-384 on invasion and migration of lung cancer SPC-A1 cells

目的观察长链非编码RNA(lncRNA)结直肠肿瘤差异表达基因(CRNDE)靶向微小RNA(miRNA,miR)-384对人肺腺癌细胞株SPC-A1细胞侵袭和迁移的影响.方法在肺癌SPC-A1细胞中转染小干扰RNA(siRNA)CRNDE,实时定量聚合酶链反应(Real-timePCR)测定干扰效果,噻唑蓝(MTT)法测定细胞增殖变化,Transwell小室测定细胞侵袭和迁移,蛋白质印迹法(Westernblot)检测细胞中基质金属蛋白酶(MMP)-9、MMP-2、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)蛋白表达水平,靶向关系预测软件预测CRNDE与miR-384有结合位点,荧光素酶报告系统鉴定靶向关系.在SPC-A1细胞中共转染siRNACRNDE和miR-384抑制物(Inhibitors),用上述方法测定miR-384Inhibitors对siRNACRNDE影响肺癌SPC-A1细胞侵袭和迁移的逆转作用.两组间比较采用t检验,多组差异比较采用单因素方差分析.结果转染siRNACRNDE后的肺癌SPC-A1细胞中CRNDE表达水平降低(1.01±0.12比0.43±0.06),细胞增殖能力降低(0.37±0.05比0.24±0.02,F=10.467,P<0.05),细胞侵袭(141.25±15.23比101.02±9.84,F=9.420,P<0.05)和迁移(188.20±16.84比142.58±11.22,F=8.345,P<0.05)数目减少,细胞中MMP-9、MMP-2、Vimentin蛋白水平下降,E-cadherin蛋白水平升高.CRNDE与miR-384有互补结合位点,CRNDE靶向负调控miR-384表达.miR-384 Inhibitors可以逆转siRNACRNDE对肺癌细胞增殖(0.26±0.03比0.35±0.04,t=3.118,P<0.05)、迁移(139.84±10.57比176.51±12.45,t=5.276,P<0.05)、侵袭(99.84±10.02比134.25±11.25,t=3.890,P<0.05)能力抑制作用和MMP-9、MMP-2、E-cadherin、Vimentin蛋白表达影响.结论下调lncRNACRNDE靶向miR-384抑制肺癌SPC-A1细胞侵袭和迁移.

Objective To investigate the effect of long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) targeting microRNA (miRNA, miR)-384 on invasion and migration of lung cancer SPC-A1 cells. MethodsSmall interfering RNA (siRNA) CRNDE was transfected into lung cancer SPC-A1 cells, and the interference effect was determined by real-time quantitative polymerase chain reaction (Real-time PCR). Cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay, cell invasion and migration were measured by Transwell chamber, and Western blotting was used to detect the expression of matrix metalloproteinase (MMP)-9, MMP-2, E-cadherin and Vimentin in cells. The targeting relationship prediction software predicts that CRNDE binds to miR-384. Luciferase reporting system was used to identify the targeting relationship. In SPC-A1 cells, siRNA CRNDE and miR-384 inhibitors were co-transfected, and the above methods were used to determine the reverse effect of miR-384 inhibitors on the invasion and migration of lung cancer SPC-A1 cells transfected with siRNA CRNDE.Comparison two groups was performed using t test, and comparison three groups was performed with one-way analysis of variance (ANOVA). ResultsThe expression of CRNDE in lung cancer SPC-A1 cells transfected with siRNA CRNDE decreased (1.01±0.12 vs. 0.43±0.06), cell proliferation decreased (0.37±0.05 vs. 0.24±0.02, F=10.467, P<0.05), the number of invasive cells (141.25±15.23 vs. 101.02±9.84, F=9.420, P<0.05) and migrating cells (188.20±16.84 vs. 142.58±11.22, F=8.345, P<0.05) decreased, the expression levels of MMP-9, MMP-2 and Vimentin proteins in cells decreased, and E-cadherin protein level increased. There were complementary binding sites between CRNDE and miR-384. CRNDE targeted and negatively regulated the expression of miR-384. The miR-384 inhibitors reversed the effects of siRNA CRNDE on proliferation (0.26±0.03 vs. 0.35±0.04, t=3.118, P<0.05), migration (139.84±10.57 vs. 176.51±12.45, t=5.276, P<0.05), invasion (99.84±10.02 vs. 134.25±11.25, t=3.890, P<0.05) and expression of MMP-9, MMP-2, E-cadherin and Vimentin in lung cancer cells. ConclusionDown-regulation of lncRNA CRNDE targeting miR-384 inhibits invasion and migration of lung cancer SPC-A1 cells.