lncRNA RP11-297P16.4促进肺腺癌细胞侵袭及迁移的机制探讨

Mechanism of lncRNARP11-297P16.4 promoting invasion and migration of lung adenocarcinoma cells

目的:探究长链非编码RNA(long non-coding RNA,lncRNA)RP11-297P16.4对人肺腺癌细胞H1299和A549侵袭及迁移的影响。方法:利用qRT-PCR检测在肺腺癌细胞H1299、A549及人正常支气管上皮细胞HBEC中RP11-297P16.4的相对表达水平;设计合成针对RP11-297P16.4的特异性shRNA,慢病毒感染建立稳定敲低lncRNA RP11-297P16.4的H1299、A549细胞;将其分为对照/H1299组、实验/H1299组、对照/A549组、实验/A549组,对照组感染shRNA-NC,实验组感染shRNA-RP11-297P16.4;qRT-PCR检测shRNA敲低效率;采用细胞划痕实验和Transwell实验检测RP11-297P16.4对肺腺癌细胞侵袭、迁移能力的影响;采用qRT-PCR和蛋白质印迹法检测基质金属蛋白酶(matrix metalloproteinase,MMP)-2和-9的mRNA和蛋白质表达水平。结果:与对照组相比,RP11-297P16.4在肺腺癌细胞中高表达,差异有统计学意义(P<0.001,P<0.01);qRT-PCR分析确定,shRNA敲低后RP11-297P16.4的mRNA表达水平显著降低(P<0.001,P<0.001);划痕实验表明24 h、48 h实验组细胞伤口愈合率均低于对照组,差异有统计学意义(P<0.05,P<0.01;P<0.001,P<0.001);Transwell小室检测结果显示,实验组细胞侵袭数量显著少于对照组,差异有统计学意义(P<0.001,P<0.001);qRT-PCR和蛋白印迹法结果显示,实验组细胞中MMP-2和MMP-9 mRNA和蛋白质表达量均低于对照组,差异具有统计学意义(均P<0.05)。结论:lncRNA RP11-297P16.4通过促进MMP-2和MMP-9 mRNA和蛋白表达来增强H1299、A549细胞的侵袭和迁移能力。

Objective:To investigate the effect of long non-coding RNA(lncRNA)RP11-297P16.4 on the invasion and migration of human lung adenocarcinoma cells of H1299 and A549.Methods:The relative expression levels of RP11-297P16.4 in H1299,A549 and human normal bronchial epithelial cells HBEC were detected by real-time fluorescence quantitative PCR.Special shRNA for RP11-297P16.4 was designed and synthesized.H1299,A549 cells with stable knockdown of lncRNA RP11-297P16.4 were established by lentivirus infection,then they were divided into control/H1299 group,experimental/H1299 group,control/A549 group and experimental/A549 group.The control group was infected with shRNA-NC,and the experimental group was infected with shRNA-RP11-297P16.4.qRT-PCR was used to detect the shRNA inhibition efficiency.Cell scratch and Transwell assays were used to establish the effect of RP11-297P16.4 on the invasion and migration of lung adenocarcinoma cells.qRT-PCR andWestern blot were performed to assay the mRNA and protein expression levels of matrix metalloproteinase(MMP)-2 and-9.Results:Compared with the control group,RP11-297P16.4 was highly expressed in lung adenocarcinoma cells,and the difference was statistically significant(P<0.001,P<0.01).qRT-PCR analysis determined that the mRNA expression level of RP11-297P16.4 was significantly reduced after shRNA knockdown(P<0.001,P<0.001).The scratch assay demonstrated that the wound healing rate of cells in the experiment group at 24 h and 48 h was lower than that in the control group,and the difference was statistically significant(P<0.05,P<0.01,P<0.001,P<0.001).Transwell experiment showed that the number of cellular invasion and migration in the experiment group was significantly less than that in the control group,and the difference was statistically significant(P<0.001,P<0.001).The results of qRT-PCR andWestern blot revealed that the mRNA and protein expression levels of MMP-2 and MMP-9 in the experiment group were lower than those in the control group,and the difference was statistically significant(all P<0.05).Conclusion:The lncRNA RP11-297P16.4 enhanced the invasive and migratory abilities of H1299 and A549 cells by promoting MMP-2 and MMP-9 expression.