Cloning of the matricellular CCN1 promoter for the molecular study in non-small-cell lung carcinomas

• Main Authors: Jeng-Jung Wang, 王正忠
• Other Authors: Shr-Jeng Leu
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/93853765781995404669

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系暨研究所 === 100 === CCN1 is a secreted matrix protein that has multiple roles in physiological and pathological conditions. The recent report shows that CCN1 can reinforce cellular senescence of skin fibroblast in the context of wound healing process. Senescence, the induction of permanent cell-cycle arrest, is an important guardian that limits cancer cell growth, thereby avoiding tumorigenesis. Based on available information, it has been proposed that CCN1 is a tumor suppressor that protects against human non-small cell lung carcinomas (NSCLCs). However, the molecular mechanism of CCN1-mediated NSCLC suppression is largely unknown. We found that CCN1 could directly induce lung cancer cell senescence. Under CCN1 treatment, NSCLC cells exhibited G1-phase block in cell cycle, along with the hypo-phosphorylation of Rb, reduction of cyclin D1, and accumulation of cell-cycle regulatory proteins, including p53 and p21. In addition, upregulation of CCN1 expression by senescent cancer cells was found in the conditions that trigger cancer senescence, including long-term treatment of low-dose TGF-?? as well as anti-cancer drugs. It is likely that the CCN1-containing microenvironment surrounding cancer cells may provide negative signals to suppress NSCLCs. We have cloned the promoter region (~ 1kb) of human CCN1 gene in order to characterize its molecular regulation under stress-induced cancer senescence. The CCN1 promoter was subcloned into the pGL3 luciferase vector. Our preliminary results showed that this reporter construct is a useful indicator of promoter activity of CCN1 gene. The mutant constructs that contain consensus sequence of DNA deletion and site-directed mutagenesis in the AP-1 and Smad-binding sites have been made. Thereby, we will further investigate the involvement of TGF-?? and JNK signaling pathways in the molecular regulation of CCN1 expression during lung tumorigenesis.