Cathepsin L Promotes Ionizing Radiation-induced Glioma Migration And Invasion Through Regulation Of CUX1/GSK-3β Pathway

Objective:The aim of this study is to preliminarily investigate the possible mechanism of cathepsin L in mediating cell invasion and migration of human glioma U251cells induced by ionizing radiation via using glioma cell lines U251 cells as model.Methods:X-ray and cathepsin L over-expression lentivirus were used to make the cathepsin L over-express in U251 cells.U251 cells were transfected with DNA and siRNA plasmids.The IP method was performed to detect the interaction between cathepsin L and CUX1 or cathepsin L and p-GSK-3β^Ser9.The migration ability of cells was measured by wound migration assay,and cell invasion was detected by transwell invasion assay.Western blot and immunofluorescence assays were used to detect the expression levels of proteins（cathepsin L,CUX1,GSK-3β）and proteins related to EMT（E-cadherin,Vimentin,N-cadherin）.FITC-Phalloidin fluorescent staining was adopted to observe the actin remodeling of U251 cells.Western blot assay was used to detect the correlation between cathepsin L and p-GSK-3β^Ser9.Results:Silencing of cathepsin L in U251 cells and X-ray treatment significantly inhibited proteolytic processing of the CUX1 transcription factor and phosphorylation of serine-9 of GSK-3β.Cathepsin L and CUX1 or cathepsin L and p-GSK-3β^Ser9 could combine with each other.Wound migration and transwell invasion assays showed that silencing of CUX1 or p-GSK-3β^Ser9 blocked X-ray or cathepsin L over-expression induced cell migration and invasion.Western blot and immunofluorescence assays showed that silencing of CUX1 or p-GSK-3β^Ser9 inhibited X-ray or cathepsin L over-expression promoted EMT.Western blot and immunohistochemistry assays revealed that cathepsin L expression had a positive correlation with p-GSK-3β^Ser9 in 13 specimens.Conclusion:Our data showed that cathepsin L activated by IR or over-expression of cathepsin L was capable of processing CUX1 to produce the physiologically relevant p110isoform so as to promote EMT.On the other hand,IR-induced EMT as well as migration and invasion are through cathepsin L mediated AKT/GSK-3β/snail pathway.