MicroRNA-608调控MIF基因影响脑胶质瘤干细胞生物学行为机制的研究

It has been reported that elevated expression of macrophage migration inhibitory factor (MIF) correlates with tumor recurrence and poor prognosis of patients with gliomas. Our previous study has shown that the expression of MIF gene and protein was significantly increased in the brain glioma stem cells (GSCs) using real-time PCR and Western blot. Recent evidence has shown that MIF may bind to CD74/CD44 receptor complex and promote migration, invasion, and proliferation in malignant gliomas by up-regulating the expression of Cdc42 gene. MicroRNA-608 (miR-608) is one of the newly discovered microRNAs, but its biological functions are not clearly understood. Only a report has shown that miR-608 could regulate the expression of CD44 gene in the human breast cancer cell line, MT-1. Our results of bioinformatic analysis have predicted that the 3'UTR of human MIF contained a region (nucleotides 73-80) with perfect complementarity with miR-608. Luciferase reporter assay revealed that miR-608 could negatively regulate the gene expression of MIF at the posttranscriptional level by the direct interaction with MIF 3'UTR target sites, and miR-608 expression was significantly down-regulated in the GSCs using end-point PCR and real-time PCR. Moreover, the Matrigel invasion assay has shown that the overexpression of miR-608 could attenuate the invasion of GSCs. Therefore, we hypothesize that low expression of miR-608 in the GSCs may relieve its suppression on the potential oncogene MIF at the posttranscriptional level and up-regulate the expression of MIF gene. MIF may bind to CD74/CD44 receptor complex and up-regulate the expression of Cdc42 gene which might activate EGF-ERK pathway and/or inhibit JNK/c-Jun pathway, and then regulate the biological behaviour of GSCs including apoptosis, proliferation, migration and invasion, etc. To verify the speculation, based on our previous study, we aim to prove the direct interaction and key binding sites between the seed sequence of miR-608 and MIF 3'UTR target sites using luciferase reporter assay. We furtherly establish GSCs models with the overexpression and low expression of miR-608 and implant tumor in nude mice to demonstrate the regulatory actions of miR-608 on the expression and function of MIF gene and the biological behaviour of GSCs in vitro and in vivo. Finally, we will investigate whether MIF could up-regulate the expression of Cdc42 gene by binding to CD74/CD44 receptor complex under the action of miR-608, and whether Cdc42 could modulate the biological behaviour of GSCs by activating EGF-ERK pathway and/or inhibiting JNK/c-Jun pathway. The results of the study may demonstrate the molecular mechanism of miR-608 mediated modulation on the biological behaviour of GSCs by down-regulating the expression of MIF gene, which will help us to reveal the pathogenesis of brain glioma and provide new therapeutic targets for malignant glioma.

巨噬细胞移动抑制因子(MIF)的表达和胶质瘤患者的复发率、生存期负相关。我们前期研究发现MIF基因在胶质瘤干细胞(GSCs)中的表达显著增加。MIF可能与CD74/CD44受体复合物结合，上调Cdc42的基因表达，促进胶质瘤侵袭。最近我们预测到MIFmRNA-3'UTR端存在miR-608结合位点；初步验证了二者结合；GSCs中miR-608表达显著下调。由此建立了在GSC中miR-608调控MIF基因功能变化进一步调控GSCs的生物学行为的工作假说。本项目拟首先验证miR-608通过"种子区"与MIF基因的直接作用和关键结合位点；进一步明确miR-608对MIF基因表达和功能的调节作用以及对GSCs的生物学行为的影响；最后研究miR-608作用下，MIF与受体复合物结合，上调Cdc42调控GSCs生物学行为的分子机制。本项目不仅能进一步揭示脑胶质瘤的发生发展机制，而且能为治疗提供新靶点。

研究证明胶质瘤干细胞(Glioma Stem Cells，GSCs)与胶质瘤的恶性生物学行为密切相关且降低放疗和化疗的疗效。许多MicroRNAs(miRNAs)在胶质瘤干细胞有异常表达，发挥致癌基因或抑癌基因的功能。miR-608研究较少且生物学行为研究的尚不清楚，巨噬细胞移动抑制因子(Macrophage migration inhibitory factor, MIF)的过表达已证实可促进胶质瘤的肿瘤复发和引起预后不良。本项目首次探讨了miR-608在脑胶质瘤干细胞中的作用及分子机制。结果表明MiR-608通过靶向调控MIF基因的表达在GSCs中发挥抑癌基因的作用。miR-608在GSCs中的表达水平显著下降，MIF基因和蛋白在GSCs中的表达水平显著增加。miR-608过表达可抑制MIF基因及蛋白的表达水平， miR-608沉默可促进MIF基因及蛋白的表达水平。双荧光素酶报告基因结果证明MIF是miR-608的靶基因。miR-608过表达可抑制脑胶质瘤干细胞的增殖、迁移、侵袭，促进其细胞凋亡。并且miR-608过表达同时MIF沉默可抑制脑胶质瘤干细胞的增殖、迁移、侵袭，促进其细胞凋亡。Western blot结果发现miR-608过表达同时MIF沉默可显著抑制脑胶质瘤干细胞中p-PI3K、p-Akt及p-JNK蛋白表达水平。综上所述，本研究首次揭示了miR-608对脑胶质瘤干细胞生物学行为的调节作用。miR-608通过下调MIF基因的表达水平，抑制PI3K/Akt和JNK信号通路，进而抑制脑胶质瘤干细胞的增殖、迁移、侵袭，并促进细胞凋亡。因此miR-608可能是脑胶质瘤治疗的一个新靶点。