miR-217靶向FOXO3影响非小细胞肺癌对吉非替尼耐药性及相关机制

Effect of miR⁃217 targeting FOXO3 on the resistance of non⁃small cell lung cancer to gefitinib and its related mechanisms

目的检测miR-217对非小细胞肺癌吉非替尼耐药性的影响,并探讨下游靶基因及相关通路。方法qRT-PCR检测人肺正常上皮细胞系BEAS-2B,NSCLC细胞系A549、HCC827、PC9、NCI-H1975及吉非替尼耐药株PC9/GR中miR-217表达量。选取PC9/GR细胞,利用转染技术构建control组、NC-mimic组、miR-217 mimic组、miR-217 mimic+si-NC组、miR-217 mimic+si-FOXO3组细胞,CCK8及克隆形成实验检测细胞增殖能力、流式细胞仪检测细胞凋亡能力,Western blot检测PI3K/AKT信号通路蛋白表达。Targetscan生物信息学网站预测miR-217下游靶基因,双荧光素酶实验检测miR-217与靶基因FOXO3相关性。结果与BEAS-2B相比,miR-217表达量在A549、HCC827、PC9、NCI-H1975细胞中显著降低(P<0.05),随着吉非替尼培养浓度增加,PC9细胞中miR-217基因表达量逐渐降低(P<0.05),并且miR-217在PC9/GR细胞中的表达低于PC9(P<0.05)。相较于control及NC-mimic组,miR-217 mimic组细胞增殖能力明显降低(P<0.05),细胞凋亡数量增多(P<0.05),p-PI3K和p-AKT蛋白表达量降低(P<0.05)。双荧光素酶报告表明FOXO3是miR-217的靶标。回复实验测得,与miR-217 mimic组及miR-217 mimic+si-NC组相比,miR-217 mimic+si-FOXO3组细胞耐药能力升高(P<0.05),增殖能力显著提升(P<0.05),细胞凋亡数量减少(P<0.05),p-PI3K和p-AKT蛋白表达量增高(P<0.05)。结论miR-217过表达逆转NSCLC细胞PC9/GR对吉非替尼的耐药性并抑制PC9/GR细胞增殖加速凋亡,这种机制可能与靶向FOXO3调控PI3K/AKT信号通路相关。

Objective To investigate the effect of miR⁃217 on gefitinib resistance in non⁃small cell lung cancer(NSCLC),and to explore the downstream target genes and related pathways.Methods qRT⁃PCR was used to detect the expression of miR⁃217 in human lung normal epithelial cell lines BEAS⁃2B,NSCLC cell lines A549,HCC827,PC9,NCI⁃H1975 and gefitinib resistant strain PC9/GR.PC9/GR cells were selected and the cells of control group,NC⁃mimic group,miR⁃217 mimic group,miR⁃217 mimic+si⁃NC group,and miR⁃217 mimic+si⁃FOXO3 group were constructed using liposome transfection technique.CCK8 and clonal formation assay were used to detect changes in cell proliferation capacity,flow cytometry was used to detect changes in cell apoptosis capacity,and western blot was used to detect protein expression related to PI3K/AKT signaling pathway.The Targetscan bioinformatics website predicted the downstream target genes of miR⁃217,and the correlation between miR⁃217 and the target gene FOXO3 was detected by dual luciferase assay.Results Compared with BEAS⁃2B cells,the expression of miR⁃217 in A549,HCC827,PC9 and NCI⁃H1975 cells was significantly decreased(P<0.05).With the increase of gefitinib concentration,the expression of miR⁃217 gene in PC9 cells was gradually decreased(P<0.05),and the expression of miR⁃217 in PC9/GR cells was lower than that in PC9(P<0.05).Compared with the control group and NC⁃mimic group,the cell proliferation capacity of miR⁃217 mimic group was significantly decreased(P<0.05),the number of apoptosis was increased(P<0.05),and the expression levels of p⁃PI3K and p⁃AKT were decreased(P<0.05).Dualluciferase reporter gene assay proved that FOXO3 is the target of miR⁃217.Compared with miR⁃217 mimic group and miR⁃217 mimic+si⁃NC group,the cell drug resistance of miR⁃217 mimic+si⁃FOXO3 group was increased(P<0.05),the proliferation ability was significantly increased(P<0.05),and the number of apoptosis was decreased(P<0.05).The expression levels of P⁃PI3K and P⁃AKT were increased(P<0.05).Conclusion Overexpression of miR⁃217 reversed the resistance of PC9/GR to gefitinib in NSCLC cells and inhibited the proliferation and accelerated apoptosis of PC9/GR cells,which may be related to the regulation of PI3K/AKT signaling pathway by targeting FOXO3.