miR-124靶向STAT3抑制胶质瘤细胞U251免疫逃逸  

miR-124 promotes immune escape of glioma cell U251 via targeting STAT3

作　　者：孙军[1] 温昌明[1] 张保朝[1] 闻公灵[1] 刘义锋[1] 汪宁[1] 刘开祥[2] 李巍[3] SUN Jun;WEN Chang-ming;ZHANG Bao-chao;WEN Gong-ling;LIU Yi-feng;WANG Ning;LIU Kai-xiang;Li Wei(Department of Neurology,Nanyang Central Hospital,Nanyang473003,China;Department of Neurology,Affiliated Hospital of Guilin Medical College,Guilin 541001,China;Department of Neurology,General Hospital of Shenyang Military Region,Shenyang 110016,China)

机构地区：[1]南阳市中心医院神经内科,南阳473003 [2]桂林医学院附属医院神经内科,桂林541001 [3]沈阳军区总医院神经内科,沈阳110016

出　　处：《现代免疫学》2020年第6期465-470,共6页Current Immunology

基　　金：国家自然科学基金青年项目(B1401097);桂林市科技开发项目(20110119-1-6)。

摘　　要：为探究miR-124在胶质瘤细胞免疫逃逸中的作用及分子机制,qPCR法检测胶质瘤患者肿瘤组织及正常组织中miR-124表达量;ELISA和qPCR检测IL-2刺激下NK细胞系(NK-92)中IFN-γ和miR-124表达量;共培养NK细胞与胶质瘤U251细胞,细胞毒实验检测NK细胞中过表达miR-124对细胞毒性及IFN-γ水平的影响;生物信息学分析、双荧光素酶报告基因实验和RNA结合蛋白免疫沉淀实验验证miR-124和信号转导及转录激活因子3(signal transduction and activator of transcription 3,STAT3)的靶向调控关系;Western blotting检测过表达和抑制miR-124表达对STAT3蛋白水平的影响;细胞毒实验检测共转染过表达pcDNA-STAT3载体对miR-124作用下NK细胞对U251细胞细胞毒性及IFN-γ水平的影响。结果显示,与正常组织相比,miR-124在患者胶质瘤组织中的表达量显著降低(P<0.05);与对照组相比,IFN-γ和miR-124在IL-2激活的NK-92细胞中表达量显著增加(P<0.05);miR-124过表达通过促进NK细胞分泌IFN-γ显著促进NK细胞细胞毒性;miR-124与STAT3存在直接靶向作用;过表达miR-124显著抑制IL-2作用下NK-92细胞STAT3蛋白水平,抑制miR-124表达则促进STAT3蛋白表达;转染pcDNA-STAT3可逆转过表达miR-124对NK细胞细胞毒性、IFN-γ分泌的影响。以上结果表明,miR-124通过靶向抑制STAT3抑制胶质瘤细胞U251免疫逃逸。To study the potential role and molecular mechanism of miR-124 in the immune escape of glioma, qPCR was used to detect the expression of miR-124 in tumor tissues and normal tissues of glioma patients. The expression levels of IFN-γ and miR-124 were examined by ELISA and qPCR in NK cell line(NK-92) activated by IL-2;NK cells were activated by IL-2 and co-cultured with glioma U251 cells. Cytotoxicity assays were used to examine the effects of overexpression of miR-124 in NK-92 cells on cytotoxicity and IFN-γ levels. Bioinformatics prediction, dual luciferase reporter and RNA-binding protein immunoprecipitation assays(RIP) were used to validate miR-124 and signal transducer and activator of transcription 3(STAT3) targeting regulatory relationships;Western blotting was used toanalyze the effect of overexpression and inhibition of miR-124 expression on STAT3 protein level. Cytotoxicity assays were used to detect the co-transfection of overexpressed STAT3 vector on NK cells cytotoxicity to U251 and IFN-γ levels under the action of miR-124. Results showed that the expression of miR-124 in patients with glioma was significantly reduced. The expressions of IFN-γ and miR-124 in IL-2 activated NK-92 cells were significantly increased(P<0.05). miR-124 overexpression significantly promoted NK cell cytotoxicity by promoting IFN-γ secretion in NK cells(P<0.05);miR-124 directly targeted STAT3;Furthermore, after transfection with pcDNA-STAT3, the effect of overexpression of miR-124 on NK cell cytotoxicity and IFN-γ secretion was reversed. These results indicate that miR-124 inhibits the immune escape of glioma cell U251 by downregulating STAT3.

关 键 词：胶质瘤 免疫逃逸 微小核糖核酸124 信号转导及转录激活因子3 

分 类 号：R739.41[医药卫生—肿瘤]