Abstract
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.
Highlights
Micro-RNAs are short RNA molecules with an established role as important epigenetic regulators of the transcriptome via recognition of base-complementary signals in the 3’ untranslated regions of target mRNAs [1,2]
Our results have suggested that miR-744 acts as a counterpart of its host gene MAP2K4, an enhancer of cancer progression [8,10]
We were further able to show that specific knock-down of these target genes impaired cellular migration similar to miR-744 overexpression. In line with these results, we found that down-regulation of miR-744 in human GBM is accompanied by a marked increase in DVL2 and TGFB1 expression levels, indicating clinical relevance of these functional networks
Summary
Micro-RNAs (miRNAs) are short RNA molecules with an established role as important epigenetic regulators of the transcriptome via recognition of base-complementary signals in the 3’ untranslated regions of target mRNAs [1,2]. The majority of human miRNA genes are located within non-coding regions of protein-coding genes [3,4]. Increasing evidence has begun to unravel the shades of a mechanism bearing an important role in the regulation of central cellular pathways, rather than just being a biological pendent of an information compression algorithm. 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 hypothesized that miR-744 regulates glioma migration by interacting with its host’s pathways
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