The effects of crmp1 gene knockout on the invasiveness and motility of lung cancer cells

• Main Authors: Yung-sheng Chang, 張永昇
• Other Authors: none
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/70305479405451372324

碩士 === 慈濟大學 === 分子生物及細胞生物研究所 === 95 === Cancer cell metastasis and neuron navigation both undergo cytoskeleton rearrangement leading to the movement. In neuron, CRMP-1 is a collapsin responsive mediator that is involved in growth cone collapse, axon guidance and F-actin depolymerization. It also has been shown that CRMP-1 could also functions as an invasion suppressor of lung cancer. However, the precise mechanism by which CRMP-1 inhibits metastasis in lung cancer cells is not clear. Previous study to unravel the role of CRMP-1 in the invasive character of lung cancer, attempts of using crmp1 mutants and knock-down to investigate its invasion suppressing function have not been successful. Therefore we opted for cellular knock-out approach. Here we developed a rAAV vector construction procedure employing fusion PCR and a single cloning step that considerably simplifies the knockout process. We found its utility by disrupting CRMP-1 gene at specific positions (exon2) within human lung cancer cells (CLI-0). And the loss of CRMP-1 resulted in CLI-0 cells changes from epithelial-like to spindle form mesenchymal like cells. CRMP1 knock-out cells have potential ability to extend like neuron and spindle-shaped morphology indicating changes in cytoskeleton organization. The results appeared to confirm the suppressor function of crmp1 in lung cancer and collaborated with previously described model in which CMRP-1-transfected lung cancer cells have fewer F-actin-staining filopodia and become more rounded. Besides, CRMP1 knock-out cells was tested by wound healing and transwell assay to show higher migration ability than wild type CL1-0 (CRMP-1 expressed) , but similar to wild type CL1-5 (no CRMP-1 expressed). These data suggested that CRMP-1 plays a pivotal role in lung cancer metastasis. We hope the CRMP-1-/- cells will be valuable cells to explore the suppressive functions of CRMP-1 on lung cancer and it will be helpful in providing an avenue for lung cancer therapy.