Small interference RNA of c98 attenuates the anti-apoptotic action of insulin-like growth factor 1 to mouse skin keratinocytes

• Main Authors: Man-Lin Tung, 董曼琳
• Other Authors: Hung-Yi Su,Ph. D.
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/24654960526818426736

碩士 === 國立屏東科技大學 === 生物科技研究所 === 95 === Insulin-like growth factor 1 (IGF-1) is a potent mitogen and plays a survival role in the skin of mammals. We have previously identified a gene, c98 (GenBank accession number:AY170344) that is induced by IGF-1 anduts nucleotide sequences are homologous to bcl-2 in the mouse skin keratinoctes(BBA 1676:127-137, 2004). The aims of this study are to further investigate the anti-apoptotic action of c98 in mediating IGF-1 receptor (IGF1-R)using RNAi (RNA interference) technology. The individual IGF-1 and IGF1-R, and their synergistic effects on the survival of skin were examined in a mouse keratinocyte cell line(XB-2)using dexamethasone (DEX) as an anti-cancer drug. IGF-1,IGF1-R, and IGF-1-IGF1-R were all found to be able to protect epidermal keratinocytes from DEX-induced apoptosis, based on the findings that after the cells were treated with DEX, DNA laddering present in the control cells but not in those treated with IGF-1, IGF1-R, and IGF-1-IGF1-R. Using a photometric enzyme-linked immunoassay(ELISA) to quantitate keratinocyte death, we found that after addition of DEX, the reduced amounts of IGF-1, IGF1-R, IGF1-IGF1-R, and c98-siRNA were 49%, 68%, 87%, and5% respectively. To assess the inhibitory role of c98 siRNA in restoring these anti-apoptotic processes, we have generated a recombinant plasmid that contains an expression vector and c98-siRNA then transfected this plasmid into keratinocytes without IGF-1 treatment. Expression of the c98-siRNA protein was found to completely (p>0.05) restore DEX-induced apoptosis after cell transfection. These results indicate that IGF-1 and its receptor play anti-apoptotic roles in DEX-induced apoptosis in epidermal keratinocytes and this, at least in part, may bemediated through c98.