Calycosin Inhibits Migration And Invasion Through Modulation Of TGF-β Mediate Mesenchymal Properties In Glioblastoma

Objective:In order to find the molecular mechanism of calycosin on inhibiting migration and invasion,we using human glioblastoma cell line U87 and U251 for our research.Methods:1 、 Human glioblastoma U87 and U251 cell were treated with different concentrations of calycosin for 24 h, MTT assay was used to evaluate the growth and proliferation of U87 and U251 cell.2 、 Human glioblastoma U87 and U251 cell were treated with different concentrations(100μM、200μM) of calycosin for 24 h,transwell migration and invasion assay was used to evaluate the migrate and invade capabilities of U87 and U251 cell;real-time quantitative fluorescent PCR was used to detect gene expression of invasion-related and migration-related molecular;western blot was used to detect protein expression of invasion-related and migration-related molecular and TGF-β.3、TGF-β plasmid was transfected into U87 cells and treated with calycosin 24 h,transwell migration 、invasion assay and western blot were used to detected whether the overexpression of TGF-β can reverse the effects of calycosin on U87 cell migration and invasion.4、5×106 U87 cells were subcutaneously implanted in the flanks of nude mice,,calycosin with PBS was injected into the xenograft tumor model in a multi-site injection manner.Tumor size and mice weight were measured every two days.Western blot was used to detect protein expression of vimentin、N-cadherin 、snail in tumor of each group.Results:1、MTT assay suggested that in 100μM, 200μM calycosin has little effect on cell proliferation and viability of glioma(P> 0.05).2、Glioblastoma was treated with three different concentrations(0μM、100μM、200μM)of calycosin for 24 hour.Transwell migration and invasion assay show thatcalycosin could weaken the ability of the cells to migrate and invade to the lower chamber, and decreased in a dose-dependent manner.(P <0.05)3、Different concentrations( 100μM、200μM) of calycosin has inhibition on the expression of vimentin,N-cadherin,snail,MMP2,MMP9 and TGF-β in U87, U251 and there is a dose-dependent.4、Overexpression TGF-β can reverse the anti-invasive effects of migration of calycosin, and reverse calycosin induced mesenchymal-related protein vimentin,N-cadherin,snail and MMP2, MMP9 expression of protein(P <0.05).5、 In vitro experiments in mice, subcutaneous tumor size in the experimental group is smaller than the control group(P <0.05), subcutaneous tumor TGF-βexpression in the experimental group is lower than the control group(P <0.05),subcutaneous tumor associated mesenchymal the marker vimentin,N-cadherin and other molecules expression in the experimental group is lower than the control group(P <0.05).Conclusion:1、 Calycosin inhibit glioblastoma cell migration and invasion in vitro and vivo.2、Calycosin inhibits the migration and invasion of glioma cells by regulating the expression of TGF-β1 target on mesenchymal-related protein and MMP2、MMP9.