circAGFG1对非小细胞肺癌生物学行为的影响机制研究

Mechanism of circAGFG1 on biological behavior of non-small cell lung cancer

目的 探讨环状RNA(circRNA)AGFG1通过靶向微小RNA(miR)-374a-5p/血管内皮生长因子C(VEGFC)轴对非小细胞肺癌(NSCLC)生物学行为的影响机制。方法 qRT-PCR检测55例NSCLC患者的癌组织和癌旁组织以及人支气管上皮细胞系(BEAS-2B)、人NSCLC细胞系(HCC827、H1975、A549、H1299)中circAGFG1、miR-374a-5p、VEGFC mRNA表达水平。以A549细胞为研究对象,设置对照组、circAGFG1小干扰RNA(si circAGFG1)组及阴性对照(si NC)组、si circAGFG1+miR-374a-5p抑制物(miR-374a-5p inhibitor)组以及si circAGFG1+抑制物对照组(inhibitor NC)组。qRT-PCR检测各组A549细胞中circAGFG1、miR-374a-5p、VEGFC mRNA表达水平;CCK-8及Transwell实验分别检测细胞活力及迁移、侵袭能力;Western blot检测增殖蛋白Ki-67、基质金属蛋白酶-9(MMP-9)以及VEGFC蛋白表达;双荧光素酶报告基因实验验证circAGFG1、VEGFC分别与miR-374a-5p的靶向关系。结果 癌组织及人NSCLC细胞系中circAGFG1、VEGFC mRNA表达水平显著增加,miR-374a-5p表达显著降低(P<0.05)。与对照组、si NC组比较,si circAGFG1组培养24 h、48 h的OD450值,迁移数,侵袭数及Ki-67、MMP-9、circAGFG1、VEGFC表达水平显著下降/减少(P<0.05),miR-374a-5p表达显著增加(P<0.05);与si circAGFG1+inhibitor NC组比较,circAGFG1+miR-374a-5p inhibitor组培养24 h、48 h的OD450值,迁移数,侵袭数及Ki-67、MMP-9、VEGFC表达水平显著增加(P<0.05),miR-374a-5p表达显著降低(P<0.05),但circAGFG1无显著变化(P>0.05)。circAGFG1、VEGFC分别与miR-374a-5p存在靶向关系。结论 干扰circAGFG1表达可通过调控miR-374a-5p/VEGFC轴抑制A549细胞的恶性生物学行为发展。

Objective To investigate the mechanism of the effect of circular RNA(circRNA)AGFG1 on the biological behavior of non-small cell lung cancer(NSCLC)by targeting microRNA(miR)-374a-5p/vascular endothelial growth factor C(VEGFC)axis.Methods The expression levels of circAGFG1,miR-374a-5p and VEGFC mRNA in tumor tissues and adjacent tissues of 55 NSCLC patients,human bronchial epithelial cell line(BEAS-2B)and human NSCLC cell line(HCC827,H1975,A549,H1299)were detected by qRT-PCR.Taking A549 cells as the research object,control group,circAGFG1 small interfering RNA(si circAGFG1)group and negative control(si NC)group,si circAGFG1+miR-374a-5p inhibitor group,and si circAGFG1+inhibitor NC group were set up.The expression levels of circAGFG1,miR-374a-5p and VEGFC mRNA of A549 cells in each group were detected by qRT-PCR;CCK-8 and Transwell assays were used to detect the cell viability,migration and invasion abilities;Western blot was used to detect the expression of proliferating protein Ki-67,matrix metalloproteinase-9(MMP-9)and VEGFC;dual luciferase gene reporter assay was used to verify the targeting relationship between circAGFG1,VEGFC and miR-374a-5p,respectively.Results The expression levels of circAGFG1 and VEGFC mRNA in cancer tissues and human NSCLC cell lines were obviously increased,while the expression of miR-374a-5p was obviously decreased(P<0.05).Compared with the control group and the si NC group,the OD450 values after 24 hours and 48 hours of culture,migration number,invasion number,and the expression levels of Ki-67,MMP-9,circAGFG1,and VEGFC in the si circAGFG1 group were obviously decreased(P<0.05),while the expression of miR-374a-5p was obviously increased(P<0.05).Compared with the si circAGFG1+inhibitor NC group,the OD450 values after 24 hours and 48 hours of culture,migration number,invasion number,and the expression levels of Ki-67,MMP-9,and VEGFC in the circAGFG1+miR-374a-5p inhibitor group were obviously increased(P<0.05),the expression of miR-374a-5p was obviously decreased(P<0.05),while circAGFG1 had no obvious change(P>0.05).circAGFG1 and VEGFC had targeting relationships with miR-374a-5p,respectively.Conclusion Interference with the expression of circAGFG1 can inhibit the development of malignant biological behavior of A549 cells by regulating miR-374a-5p/VEGFC axis.