目的 探讨miR-124*和laminin-8 β1链在不同恶性程度人脑胶质瘤中的表达,分析两者间潜在的表达调控关系. 方法 Western blotting、实时荧光定量PCR分别检测南方医科大学珠江医院神经外科自201 1年1月至2012年12月间手术切除并经病理证实的30例人脑胶质瘤标本和2例正常脑组织标本中laminin-8 β1链蛋白、miR-124*的表达.构建质粒载体psiCHECKTM-2-野生型(WT) laminin β1链3'非翻译区(3'UTR)和psiCHECKTM-2-突变型(MUT)laminin β1链3'UTR,分别同miR-124*模拟物、miRNA模拟物阴性对照;miR-124*抑制剂、miRNA抑制剂阴性对照共转染至U87细胞中,转染空载体psiCHECKTM-2作为空白对照.转染48 h后采用双荧光素酶报告检测系统检测各组细胞的荧光强度.nuu裸鼠皮下注射U87细胞构建胶质瘤动物模型,miR-124*模拟物原位注射治疗3周后Western blotting检测肿瘤中laminin-8 β1链蛋白表达,免疫组化染色观察血管内皮细胞标记物CD31的表达. 结果 Western blotting显示Laminin-8蛋白在高级别胶质瘤(WHOⅢ/Ⅳ级)中呈现高表达,而在低级别胶质瘤(WHO Ⅰ/Ⅱ级)中表达不明显;实时荧光定量PCR显示miR-124*在正常脑组织、WHO Ⅰ级、Ⅱ级、高级别胶质瘤中表达依次降低,差异有统计学意义(P＜0.05).双荧光素酶报告实验显示,与psiCHECKTM-2-WTlaminin β1链3'UTR质粒共转染时,miR-124*模拟物组荧光强度(2.13±0.25)明显弱于miRNA模拟物阴性对照组(2.71±0.08),而miR-124*抑制剂组(3.18±0.22)明显强于miRNA抑制剂阴性对照组(2.70±0.17),差异有统计学意义(P＜0.05).动物模型实验结果提示miR-124*治疗组较对照组肿瘤laminin-8 β1链及CD31表达均明显降低. 结论 miR-124*通过负调控laminin-8 β1链的表达影响胶质瘤的恶性程度,可作为诊断治疗胶质瘤的靶基因.%Objective To explore the expressions ofmiR-124* and laminin-8 in different grades of human gliomas,and analyze the potential regulatory relationship between them.Methods Thirty human glioma specimens of different stages and two healthy brain tissues,collected in our hospital from January 2011 to December 2012,were used in our study; Western blotting was performed to detect the laminin-8 and miR-124* protein expressions; plasmid vectors carried psiCHECKTM-2-wild-type (WT) laminin β1 chain 3 'untranslated region (3'UTR) and psiCHECKTM-2-mutant (MUT) laminin β1 chain 3'UTR were established,and they were,respectively,co-transfected with the miR-124* mimics,negative control ofmiRNA mimics,miR-124* inhibitors,negative control ofmiRNA inhibitors into the U87 cells;besides,cotransfection with psiCHECKTM-2 into U87 cells was used as blank control group; the fluorescence intensity of each group was determined by dual-luciferase assay 48 h after the cotransfection.Dual-luciferase assay was developed to verify the regulatory effects ofmiR-124* on laminin-8.The nuu nude mice were given subcutaneous injection of U87 cells to establish giloma animal models; the expression of β1 chain-containing laminin-8 protein was detected in gliomas by Western blotting; the expression of vascular endothelial cell marker CD31 was observed by immunohistochemical staining.Results Western blotting showed high expressions of laminin-8 protein in high-grade gliomas (WHO grade Ⅲ/ⅣV),but low expressions in low-grade gliomas (WHO grade Ⅰ/Ⅱ); real-time fluorescent quantitative PCR showed that the expression of miR-124* in high-grade gliomas was significantly lower than that in low-grade gliomas.Dual-luciferase assay confirmed that fluorescence intensity of U87 cells carried psiCHECKTM-2-WT laminin β1 chain 3'UTR and miR-124* mimics (2.13±0.25) was significantly weaker than that of cells carried psiCHECKTM-2-WT laminin β1 chain 3'UTR and negative control of miRNA mimics (2.71±0.08,P＜0.05); that of those carried psiCHECKTM-2-MUT laminin β1 chain 3'UTR and miR-124* inhibitors (3.18 ±0.22) was significantly stronger than those carried,psiCHECKTM-2-MUT laminin β1 chain 3'UTR and negative control of miRNA inhibitors (2.70±0.17,P＜0.05).The animal model results indicated the laminin-8 and CD31 expressions in miR-124* treatment group were obviously lower than those in control group.Conclusion MiR-124*,which can affect the malignant degrees of human gliomas through playing its negative regulation role in laminin-8 expression,maybe a target gene for the diagnosis and therapy of gliomas.
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