The Effects Of Over-expression Of Cathepsin L On Cell Invasion And Migration In Human Brain Glioma U251 Cells

Aim:In this study, we would investigate the effect and the mechanisms of the over-expression of cathepsin L in the human brain gliomain vasion and migration.Methods:X-ray and cathepsin L over-expression lentivirus was used to make the CTSL over-express in U251 cells. U251 cells migration induced by CTSL over-expression was measured in vitro by wound healing assayand cell invasion was detected by transwell invasion assay. Colony formation approach was used to measure the multiplication capacity of U251 cells. Western blotting and immunofluorescence assay were used to detect the expression levels of the proteins (cathepsin L, E-cadherin, N-cadherin, vimentin) related to the invasion in U251 cells. Activation of RhoA and CDC42 was measured by G-LISA RhoA/CDC42 Activation Assay Kit. FITC-Phalloidin fluorescent staining and immunofluorescence assay were adopted to observe the actin remodeling of U251 cells. Western blotting and immunofluorescence assay were used to detect the expression levels of the proteins (cathepsin L, E-cadherin, N-cadherin, vimentin, snail) in 13 clinical human glioma spacimens.Results:X-ray irradiation and cathepsin L over-expression lentivirus both improve the potential of human glioma U251 cells for cellular migration and invasion. Over-expression of cathepsin L promotes proliferation of U251 cells. Proteins related to epithelial mesenchymal transition in U251 cells including N-cadherin, vimentin and snail were proved to be increased by western blotting and immunofluorescence assay. And E-cadherin was proved to be decreased. G-LISA Rho/CDC42 Activation Assay revealed that RhoA and CDC42 activition can be increased by the upregulation of cathepsin L. FITC-Phalloidin fluorescent staining and immunofluorescence assay indicated that the upregulation of cathepsin L in U251 cells was associated with increase in cell spreading, actin stress fiber formation and actin polymerization. Western blotting revealed that cathepsin L expression had a negative correlation with E-cadherin but positive with mesenchymal marker and snail in 9 of 13 specimens.Conclusion:Our data show that cathepsin L is an important protein which mediates the cell invasion of human glioma U251 cells, and its molecular mechanism may involve regulation of the protein expression of cytoskeletal protein and proteins related to epithelial mesenchymal transition (EMT)...