The Effects And Mechanisms Of Milk Fat Globule Epidermal Growth Factor 8(MFG-E8) On Glioma In The Tumor Microenvironment

Objective: To study the effects of milk fat globule epidermal growth factor 8 protein(MFG-E8) on glioma and the underlying mechanisms. To explore effects of MFG-E8 on microglia polarization in tumor microenvironment and quest its potential molecular mechanisms.Methods: The stable MFG-E8 known-down, GFP expressed GL261 glioma cell line(GL261-GFP-sh MFG-E8) was established by the GFP/MFG-E8 sh RNA recombinant lentivirus. The interference efficiency of recombinant lentivirus in GL261 glioma cells were evaluated by RT-PCR and ELISA. The proliferative ability of GL261-GFP-sh MFG-E8 cells were measured by MTT, cell counting and colony-formation assay. The migration ability of glioma cells were determined by x CELLigence Real-time Cell Analyzer and cell wound scratch assay. The drug sensitivity of glioma cells to BCNU was determined by MTT assay. The expression of survival-related protein was measured by western blots. The tumor development and procession, including tumor size, survival rate were evaluated in mouse GL261-GFP glioma model in vivo. Tumor-associated microglia infiltration and polarization were assessed by immunohistochemistry and flow cytometry.Results: Knockdown of MFG-E8 in GL261 glioma cells decreased glioma cell proliferation and migration and enhanced BCNU drug sensitivity. Knockdown of MFG-E8 in GL261 glioma cells decreased phosphorylation of FAK, AKT and ERK. Knockdown of MFG-E8 in GL261 glioma cells reduced phosphorylation of STAT3 and the expression of Cyclin D1. In GL261-GFP murine glioma model, knockdown of MFG-E8 in GL261 glioma cells reduced tumor development and procession, improved individual survival rate and decreased CD11b+ cells infiltration and M2 type marker CD206 expression.Conclusion: Knockdown of MFG-E8 by sh RNA in GL261 glioma cells significantly decreased cell proliferation and migration and enhanced BCNU sensitivity, which may be attributed to down regulation of STAT3 signaling pathway. In GL261-GFP murine glioma model, knockdown of MFG-E8 significantly decreased tumor size, which was accompanied by reduced CD11b+ cells infiltration and M2 type marker CD206 expression.