miR-224靶向抑制ECRG4表达对非小细胞肺癌细胞增殖、迁移和侵袭的影响

Effects of miR-224 targeted inhibition of ECRG4 expression on the proliferation,migration and invasion of human non-small cell lung cancer cells

目的研究微小RNA-224(miR-224)及其靶基因食管癌相关基因4(ECRG4)在非小细胞肺癌(NSCLC)中的表达和对A549细胞增殖、迁移、侵袭的影响。方法利用原位杂交、荧光定量PCR、免疫组织化学和Western blot检测NSCLC临床标本和细胞系中miR-224和ECRG4的表达水平;通过microRNA.org网站预测miR-224与ECRG4的靶向关系,并行双荧光素酶报告基因实验和Western blot进行验证;分析抑制miR-224及联合抑制ECRG4对A549细胞增殖、迁移和侵袭能力的影响。结果NSCLC癌组织中miR-224表达水平明显高于癌旁组织,ECRG4 mRNA和ECRG4蛋白表达水平均明显低于癌旁组织,差异有统计学意义(P<0.05)。与健康人肺支气管上皮细胞系HBE相比,NSCLC细胞系L78、A549、H460中miR-224表达水平明显升高,ECRG4 mRNA和ECRG4蛋白表达水平明显降低,差异有统计学意义(P<0.05)。经microRNA.org网站预测、双荧光素酶报告基因实验和Western blot证实,ECRG4是miR-224的靶基因。抑制miR-224可抑制A549细胞增殖、迁移和侵袭(P<0.05),而联合抑制ECRG4则可逆转抑制miR-224对A549细胞的抑制作用(P<0.05)。结论miR-224可通过靶向ECRG4促进NSCLC细胞A549增殖、迁移和侵袭,提示miR-224和ECRG4可能成为诊断和治疗NSCLC的靶点。

Objective To study the expression of microRNA-224(miR-224)and its target gene esophageal cancer-related gene 4(ECRG4)in non-small cell lung cancer(NSCLC),and their effects on the proliferation,migration and invasion of A549 cells.Methods Fluorescence in situ hybridization,fluorescence quantitative PCR,immunohistochemistry and Western blot were used to detect the expression levels of miR-224 and ECRG4 in clinical specimens and cell lines of NSCLC.MicroRNA.org was used to predict the targeted relationship between miR-224 and ECRG4,and double luciferase reporter gene experiment and Western blot were used for verification.The effects of miR-224 and ECRG4 on proliferation,migration and invasion of A549 cells were analyzed.Results The expression level of miR-224 in NSCLC cancer tissues was significantly higher than that in adjacent tissues,and the expression levels of ECRG4 mRNA and ECRG4 protein were significantly lower than those in adjacent tissues,the difference was statistically significant(P<0.05).Compared with healthy human lung bronchial epithelial cell line HBE,the expression levels of miR-224 in the NSCLC cell lines L78,A549 and H460 were significantly increased,and the expression levels of ECRG4 mRNA and ECRG4 protein were significantly reduced,the difference was statistically significant(P<0.05).Predicted by the microRNA.org website,double luciferase reporter gene experiment and Western blot confirmed that ECRG4 was the target gene of miR-224.Inhibition of miR-224 inhibited proliferation,migration and invasion of A549 cells(P<0.05),and combined inhibition of ECRG4 could reverse the inhibitory effect of miR-224 on A549 cells(P<0.05).Conclusion miR-224 can promote the proliferation,migration and invasion of NSCLC A549 cells by targeting ECRG4,suggesting that miR-224 and ECRG4 may be the targets for the diagnosis and treatment of NSCLC.