miR-217靶向调控ERK2表达对非小细胞肺癌细胞活性和免疫逃逸的影响及机制研究

Effect and mechanism of miR-217 targeting ERK2 expression on activity and immune escape of non-small cell lung cancer cells

目的:研究miR-217靶向调控细胞外信号调节激酶2(ERK2)表达对非小细胞肺癌(NSCLC)细胞活性和免疫逃逸的影响及作用机制。方法:qRT-PCR检测miR-217与ERK2 mRNA在NSCLC组织、癌旁组织及HLF-1、A549、HCC827细胞株中的表达量,分析不同miR-217表达量NSCLC患者的预后生存状态。使用生物信息学、双荧光素酶基因报告实验分析miR-217与ERK2的靶向关系。培养NSCLC A549细胞,分为NC组、miR-217 inhibitor组、miR-217 mimic组、miR-217 mimic+ERK2NC组、miR-217 mimic+ERK2组,除NC组不做任何处理外,其余各组均转染对应质粒,分析各组A549细胞增殖活性、免疫逃逸状况,并明确作用机制。结果:与癌旁组织比较,NSCLC组织miR-217表达量降低,ERK2 mRNA表达量升高(P<0.05)。与肺成纤维细胞HLF-1细胞株比较,NSCLC细胞A549、HCC827细胞株miR-217表达量降低,ERK2 mRNA表达量升高(P<0.05)。基于Kaplan-Meier Plotter数据库分析miR-217与NSCLC患者预后的关系,结果表明miR-217低表达与患者预后不良有关(HR=0.90,P=0.033)。双荧光素报告基因显示miR-217与ERK2之间的3'UTR区域间存在匹配序列,miR-217 mimic片段可抑制ERK2-WT信号,但对ERK2-MUT无影响。与NC组比较,miR-217 inhibitor组细胞增殖活性、PD-L1、PD-L2 mRNA表达量升高,CD8+T细胞活力降低,miR-217 mimic组细胞增殖活性、PD-L1、PD-L2 mRNA表达量降低,CD8+T细胞活力升高(P<0.05)。与miR-217 mimic组比较,miR-217 mimic+ERK2 NC组细胞增殖活性、CD8+T细胞活力、PD-L1、PD-L2 mRNA表达量无明显改变(P>0.05),miR-217 mimic+ERK2组细胞增殖活性、PD-L1、PD-L2 mRNA表达量升高,CD8+T细胞活力降低(P<0.05)。结论:miR-217过表达可降低NSCLC细胞A549活性,抑制PD-L1表达,激活肿瘤微环境中的CD8+T细胞,进而抑制免疫逃逸,其可能通过靶向调控ERK2发挥作用。

Objective:To investigate the effect and mechanism of miR-217 targeted regulation of extracellular signal-regulated kinase 2(ERK2)expression on activity and immune escape of non-small cell lung cancer cells(NSCLC).Methods:qRT-PCR was used to detect expression levels of miR-217 and ERK2 mRNA in NSCLC tissues,adjacent tissues,and HLF-1,A549 and HCC827 cell lines.Analyzed prognosis and survival status of NSCLC patients with different miR-217 expression level.Bioinformatics and dual luciferase gene reporting experiments were used to analyze the targeting relationship between miR-217 and ERK2.Cultivated NSCLC A549 cells and divided them into NC group,miR-217 inhibitor group,miR-217 mimic group,miR-217 mimic+ERK2 NC group and miR-217 mimic+ERK2 group.Except for the NC group without any treatment,all other groups were transfected with corresponding plasmids to analyze the proliferation activity and immune escape status of A549 cells in each group,and clarified the mechanism of action.Results:Compared with adjacent tissues,expression of miR-217 in NSCLC tissue was decreased,while expression of ERK2 mRNA was increased(P<0.05).Compared with human normal lung fibroblast HLF-1 cell lines,expression of miR-217 in NSCLC cell lines A549 and HCC827 were decreased,while expression of ERK2 mRNA was increased(P<0.05).Analysis of the relationship between miR-217 and prognosis of NSCLC patients based on Kaplan-Meier Plotter database showed that low expression of miR-217 was associated with poor prognosis of patients(HR=0.90,P=0.033).Dual fluorescein reporter genes showed matching sequences between the 3'UTR regions of miR-217 and ERK2.miR-217 mimic fragment could inhibit ERK2-WT signal,but had no effect on ERK2-MUT.Compared with NC group,cell proliferation activity,PD-L1 and PD-L2 mRNA expression levels of miR-217 inhibitor group were in creased,while CD8+T cell activity was decreased,and cell proliferation activity,PD-L1 and PD-L2 mRNA expression levels of miR-217 mimic group were decreased,while CD8+T cell activity was increased(P<0.05).Compared with miR-217 mimic group,cell proliferation activity,CD8+T cell activity,PD-L1 and PD-L2 mRNA expression levels of miR-217 mimic+ERK2 NC group had no significant changes(P>0.05),cell proliferation activity,PD-L1 and PD-L2 mRNA expression levels of miR-217 mimic+ERK2 group were increased,while CD8+T cell activity was decreased(P<0.05).Conclusion:Overexpression of miR-217 can reduce the activity of NSCLC cell A549,inhibit the expression of PD-L1,activate CD8+T cells in tumor microenvironment,and then inhibit immune escape,which may play a role by targeting ERK2.