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Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy
Published on: October 4, 2019
Intronic miR-744 Inhibits Glioblastoma Migration by Functionally Antagonizing Its Host Gene MAP2K4
Max Hübner1,2, Christian Ludwig Hinske3, David Effinger4,5
1Department of Anesthesiology, University Hospital, LMU Munich, 81377 Munich, Germany. max.huebner@med.uni-muenchen.de.
Background:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
Insights
MicroRNA-744 (miR-744), found in the MAP2K4 gene, restrains glioma cell migration. Reduced miR-744 in glioblastoma (GBM) correlates with increased invasiveness, suggesting miR-744 re-expression could be a therapeutic strategy.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Mitogen-Activated Protein Kinase Kinase 4 (MAP2K4) is crucial in the pro-invasive MAPK pathway.
- Intronic microRNAs (miRNAs) can regulate their host genes' functions, forming complex regulatory networks.
- This study investigates the role of miR-744, located in the MAP2K4 intron, in regulating glioma cell migration.
Purpose of the Study:
- To determine if miR-744 regulates glioma cell migration.
- To elucidate the molecular mechanisms by which miR-744 affects glioma cell invasiveness.
- To explore the potential of miR-744 as a therapeutic target for glioma.
Main Methods:
- Overexpression of miR-744 in glioblastoma (GBM) and cell lines (U87, T98G).
- Assessment of cell mobility using migration and Boyden chamber assays.
- Quantification of protein and mRNA expression (SDS-PAGE, qRT-PCR) and analysis of gene target interactions (luciferase reporter assays).
Main Results:
- miR-744 levels were significantly decreased in GBM samples and cell lines compared to normal brain tissue.
- miR-744 re-expression in GBM cells reduced cell mobility and focal growth.
- miR-744 directly targets TGFB1 and DVL2, key pro-invasive factors, and mitigates MAP2K4, SMAD, and beta-Catenin signaling pathways.
Conclusions:
- miR-744 functions as an intrinsic suppressor of its host gene, MAP2K4.
- By targeting multiple signaling pathways, miR-744 inhibits glioma cell migration and invasiveness.
- Restoration of miR-744 levels in glioma may represent a novel therapeutic approach to reduce tumor invasiveness.
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