Abstract
Glioma tumors constitute a significant portion of microglial cells, which are known to support tumor progression. The present study demonstrates that transforming growth factor-β (TGFβ) signaling pathway in microglia in a glioma environment is involved in tumor progression and pathogenesis. It has been shown that the TGFβ level is elevated in higher grades of gliomas and its signaling pathway regulates tumor progression through phosphorylation of SMAD2 and SMAD3, which form a complex with SMAD4 to regulate target gene transcription. In an in vitro cell line-based model increased protein levels of pSMAD2/3, total SMAD2/3 and SMAD4 were observed in murine BV2 microglia cultured in glioma conditioned medium (GCM), indicative of the activated TGFβ signaling pathway in microglia associated with glioma environment. Immunofluorescence labeling further revealed the expression of SMAD4 in microglial and non-microglial cells of human glioblastomas tissue in vivo. Functional analysis through shRNA-mediated stable knockdown of SMAD4 in microglia revealed the downregulation of the expression of matrix metalloproteinase 9 (MMP9), which has been shown to be involved in tumor progression and cell migration. Further, knockdown of SMAD4 in microglia decreased the migration of microglial cells towards GCM, indicating that SMAD4 promotes microglial migration in glioma environment. In addition, SMAD4 has been shown to be post-transcriptionally regulated by microRNA-146a, which was downregulated in microglia treated with GCM. Overexpression of miR-146a resulted in decreased expression of SMAD4 together with tumor supportive gene MMP9 in microglia, and subsequently suppressed microglial migration towards GCM, possibly through regulation of SMAD4. On the other hand, the cell viability assay revealed decreased viability of glioma cells when they were treated with conditioned medium derived from SMAD4 knockdown microglia or miR-146a overexpressed microglia as compared to glioma cells treated with the medium from control microglial cells. Taken together, the present study suggests that microglial SMAD4 which is epigenetically regulated by miR-146a promotes microglial migration in gliomas and glioma cell viability.
Highlights
Gliomas are malignant brain tumors with a wide range of clinical features
The present study demonstrates that transforming growth factor-β (TGFβ) signaling pathway in microglia in a glioma environment is involved in tumor progression and pathogenesis
Immunocytochemistry revealed increased co-localization of the SMAD4 and total SMAD2/3 in microglia treated with glioma conditioned medium (GCM) as compared to control cells (Figure 1E), suggesting that the TGFβ pathway is activated in GCMtreated microglia
Summary
Gliomas are malignant brain tumors with a wide range of clinical features. Gliomas arise from the glial cells of the brain, progressing through the benign stage (WHO Grade I) to highly malignant (WHO Grades II to IV) stages. Microglial function in glioma tumors is an alternative form of activation wherein microglia secrete cytokines and chemokines that are gliomagenic and support the growth of the tumor [11, 12]. Recent studies suggest that tumor-associated microglia express genes that are distinct from either activation state [13, 14], emphasizing the complex nature of tumor-associated microglia and its roles in a glioma microenvironment. This tumorigenic nature of microglia in glioma tumors may be attributed to molecular and epigenetic pathways that are altered by signaling molecules released from cancerous cells in the microenvironment
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