Hübner Max, Hinske Christian Ludwig, Effinger David, Wu Tingting, Thon Niklas, Kreth Friedrich-Wilhelm, Kreth Simone
Department of Anesthesiology, University Hospital, LMU Munich, 81377 Munich, Germany.
Walter-Brendel Center of Experimental Medicine, Faculty of Medicine, LMU Munich, 81377 Munich, Germany.
Cancers (Basel). 2018 Oct 25;10(11):400. doi: 10.3390/cancers10110400.
The second intron of Mitogen-Activated Protein Kinase Kinase 4 (MAP2K4), an important hub in the pro-invasive MAPK pathway, harbors miR-744. There is accumulating evidence that intronic micro-RNAs (miRNAs) are capable of either supporting or restraining functional pathways of their host genes, thereby creating intricate regulative networks. We thus hypothesized that miR-744 regulates glioma migration by interacting with its host's pathways.
Patients' tumor specimens were obtained stereotactically. MiR-744 was overexpressed in U87, T98G, and primary glioblastoma (GBM) cell lines. Cell mobility was studied using migration and Boyden chamber assays. Protein and mRNA expression was quantified by SDS-PAGE and qRT-PCR. Interactions of miR-744 and 3'UTRs were analyzed by luciferase reporter assays, and SMAD2/3, p38, and beta-Catenin activities by TOP/FOPflash reporter gene assays.
As compared to a normal brain, miR-744 levels were dramatically decreased in GBM samples and in primary GBM cell lines. Astrocytoma WHO grade II/III exhibited intermediate expression levels. Re-expression of miR-744 in U87, T98G, and primary GBM cell lines induced focal growth and impaired cell mobility. Luciferase activity of 3'UTR reporter constructs revealed the pro-invasive factors TGFB1 and DVL2 as direct targets of miR-744. Re-expression of miR-744 reduced levels of TGFB1, DVL2, and the host MAP2K4, and mitigated activity of TGFB1 and DVL2 downstream targets SMAD2/3 and beta-Catenin. TGFB1 knock-down repressed MAP2K4 expression.
MiR-744 acts as an intrinsic brake on its host. It impedes MAP2K4 functional pathways through simultaneously targeting SMAD-, beta-Catenin, and MAPK signaling networks, thereby strongly mitigating pro-migratory effects of MAP2K4. MiR-744 is strongly repressed in glioma, and its re-expression might attenuate tumor invasiveness.
丝裂原活化蛋白激酶激酶4（MAP2K4）的第二个内含子是促侵袭性MAPK途径中的一个重要枢纽，其中含有miR-744。越来越多的证据表明，内含子微小RNA（miRNA）能够支持或抑制其宿主基因的功能途径，从而形成复杂的调控网络。因此，我们推测miR-744通过与其宿主途径相互作用来调节胶质瘤的迁移。
通过立体定向获取患者的肿瘤标本。在U87、T98G和原发性胶质母细胞瘤（GBM）细胞系中过表达miR-744。使用迁移和博伊登小室试验研究细胞迁移能力。通过SDS-PAGE和qRT-PCR定量蛋白质和mRNA表达。通过荧光素酶报告基因试验分析miR-744与3'UTR的相互作用，并通过TOP/FOPflash报告基因试验分析SMAD2/3、p38和β-连环蛋白的活性。
与正常脑相比，GBM样本和原发性GBM细胞系中miR-744水平显著降低。WHO II/III级星形细胞瘤表现出中等表达水平。在U87、T98G和原发性GBM细胞系中重新表达miR-744可诱导局灶性生长并损害细胞迁移能力。3'UTR报告基因构建体的荧光素酶活性显示促侵袭因子TGFB1和DVL2是miR-744的直接靶标。重新表达miR-744可降低TGFB1、DVL2和宿主MAP2K4的水平，并减轻TGFB1和DVL2下游靶标SMAD2/3和β-连环蛋白的活性。敲低TGFB1可抑制MAP2K4表达。
miR-744对其宿主起到内在制动作用。它通过同时靶向SMAD、β-连环蛋白和MAPK信号网络来阻碍MAP2K4功能途径，从而强烈减轻MAP2K4的促迁移作用。miR-744在胶质瘤中受到强烈抑制，其重新表达可能会减弱肿瘤的侵袭性。
