Characterizing functions of small GTP-binding protein regulators in U251 cells

• Main Authors: Kun-LinLin, 林昆霖
• Other Authors: Chun-Chun Li
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/47b6v3

碩士 === 國立成功大學 === 生命科學系 === 104 === Glioblastoma, is the most malignant and aggressive primary brain tumor in humans. Hypoxia in glioblastoma and its microenvironment is associated with the tumor growth, progression and resistance to conventional therapy of cancers. The amount of invasionrelated molecules altered by the increase of HIF-1α expression under hypoxic stress is an essential event in the activation of glioma cell motility. Several reports indicated that hypoxia is also a well-known inducer of the epithelial to mesenchymal transition (EMT) program in many cancers, include glioblastoma. Brefeldin A-inhibited guanine nucleotide-exchange protein 1 and 2 (BIG1 and BIG2) are well known to activate, via its ~200-amino-acid Sec7 domain, the replacement of ADP-ribosylation factors (ARFs)-bound GDP with GTP to regulate membrane transport, protein trafficking and cytoskeleton remodeling in eukaryotic cells. Recent studies showed that BIG1 and BIG2 are implicated in the regulation of cell polarization and actin dynamics for cell migration. However, little is known about the role of BIG1 and BIG2 in glioblastoma and the regulation of BIGs expression in glioma cells under oxidative stress condition. In this study, we found CoCl2-induced oxidative stress influence cell morphology, affect the expression of mRNA and protein levels of epithelial-mesenchymal transition (EMT) markers, change the mRNA expression of cell proliferation markers and elevate the pAKT protein level in glioblastoma U251 cells. Protein expression levels of BIG1 and BIG2 in U251 cells after CoCl2 treatment were determined by Western blotting. We also used siRNA and shRNA that specific targeting BIG1 and BIG2 to deplete expression of BIG1 and/or BIG2 in U251 cells. Effects of BIG1 and/or BIG2 depletion on U251 cell morphology, cell proliferation, and signaling pathways were examined. The preliminary results obtained here will provide us useful information for further studies of roles of BIG1 and BIG2 in tumor growth and progression.