MUC4and FOXC2Promote Glioblastoma Proliferation And Invasion Effect And Its Molecular Mechanism

Part1FOXC2often overexpressed in glioblastoma enhances proliferation andinvasion in glioblastoma cellsObjective: To reveal the FOXC2expression in human GBM cells and the roleFOXC2played in GBM cells proliferation and invasionMethods: The FOXC2expressions in normal brain samples, GBM samples and allthe six GBM cell lines were determined by RT-PCR, immunohistochemistry andWestern blot. Construct the FOXC2-overexpressing SNB19-FOXC2cells, FOXC2expression silencing T98G-shFOXC2cells and EGFR expression silencingSNB19-FOXC2-shEGFR cells through gene transfection and shRNA interference. Theeffect of FOXC2on the proliferation ability of GBM was measured by MTT and colonyformation assays. Determine the modulatory effect of FOXC2on the migratory andinvasive capacities of SNB19-FOXC2cells by wound-healing assay and Boyden’schamber assay. Determine role FOXC2played on the expression of EGFR by RT-PCR,Western blot and immunohistochemistry.Results: The RT-PCR and Western blot results of brain samples indicated thatFOXC2expressed more in GBM brain samples than in normal brain samples as well asimmunohistochemistry results. The FOXC2expression of all six GBM cell linesindicated maximum expression in T98G cell line whereas the minimum in the SNB19cell line. So we construct the FOXC2overexpression SNB19-FOXC2cells and theFOXC2silencing T98G-shFOXC2cells. The MTT and colony formation assays resultsindicated that the cell proliferation of SNB19-FOXC2cells significantly increasedcompared with vector-only controls and SNB19-FOXC2cells generated a greaternumber of colonies and formed significantly larger colonies than vector-only controls.The wound-healing assay indicated SNB19-FOXC2cells showed significantly fasterclosure of wound area than the respective controls and the result was further confirmedby Boyden’s chamber assay and SNB19-FOXC2cells showed more than twofold cellmigration through Transwell membranes than the controls cells after24h of incubation. And the migratory and invasive capacities of T98G cells reduced after FOXC2silencing.We observed reduced proliferation, migratory, and invasive capacities after EGFRsilencing in SNB19-FOXC2cells and the immunohistochemistry result indicated ahighly positive correlation was found between FOXC2and EGFR expression.Conclusion:(1) FOXC2expressed significantly in GBM cells.(2) FOXC2plays akey role in migration, invasion, and proliferation capacities of GBM cells.(3) FOXC2mediates the EGFR expression in GBM cells.(4) EGFR could mediate FOXC2-inducedmigration, invasion, and proliferation capacities of GBM cells.Part2MUC4modulates human GBM cells proliferation and invasion byup-regulating EGFR expressionObjective: To reveal the MUC4expression in human GBM cells and the effectof MUC4to the GBM cells proliferation and invasionMethods: The MUC4expressions in normal brain samples, GBM brain samplesand six GBM cell lines were determined by RT-PCR, immunohistochemistry andWestern blot. Construct the MUC4-overexpressing GBM cells, MUC4expressionsilencing T98G cells and EGFR expression silencing SNB19-MUC4cells through genetransfection and shRNA interference. The effect of MUC4on the proliferation ability ofGBM was measured by MTT and colony formation assays. Determine the modulatoryeffect of MUC4on the migratory and invasive capacities of GBM cells bywound-healing assay and Boyden’s chamber assay. Determine the effect of MUC4onthe expression of EGFR by RT-PCR, Western blot and immunohistochemistry.Results: The RT-PCR results of brain samples indicated that MUC4expressedmore in GBM brain samples than in normal brain samples as well asimmunohistochemistry results. The MUC4expression of all six GBM cell lines weremeasured by RT-PCR and Western blot and we found the maximum expression inT98G cell line whereas the minimum in the SNB19cell line. So we construct theMUC4overexpression SNB19cell line and the MUC4silencing T98G cell line. TheMTT and colony formation assays results indicated that the cell proliferation of SNB19 MUC4cells significantly increased compared with vector-only controls andMUC4-overexpressing cells generated a greater number of colonies and formedsignificantly larger colonies than vector-only controls. The wound-healing assayindicated SNB19-MUC4cells showed significantly faster closure of wound area thanthe respective controls and the result was further confirmed by Boyden’s chamber assayand SNB19-MUC4cells showed more than threefold cell migration through Transwellmembranes than the controls cells after24h of incubation. And the migratory andinvasive capacities of T98G cells reduced after MUC4silencing. We observed reducedproliferation, migratory, and invasive capacities after EGFR knockdown inSNB19-MUC4cells and the immunohistochemistry result indicated a highly positivecorrelation was found between MUC4and EGFR expression.Conclusion:(1) MUC4expressed significantly in GBM cells and inducedmigration, invasion, and proliferation capacities of GBM cells.(2) MUC4mediated theEGFR expression in GBM cells and EGFR could mediate MUC4-induced migration,invasion, and proliferation capacities in GBM cells.