Characterizes the effects of Klf10/Sei-1 modulation in pancreatic cancer cell and gene-deficient mice

• Main Authors: Min-LuWu, 吳忞儒
• Other Authors: Hao-Wen Wang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/85795121207682181050

碩士 === 國立成功大學 === 生命科學系碩博士班 === 101 === Klf10 is a group of Krüppel-like transcription factors which generally bind to the promoter of target genes through the zinc-finger structure, and regulate their transcription. Previous study has indicated that increased intracellular levels of Klf10 mimic the anti-proliferative and apoptotic effects of TGF-β1 on epithelial cell growth, suggesting that Klf10 is an important factor for mediating TGF-β1 signaling. From ChIP-chip, which have been adopted to elucidate the novel target genes and signaling cascades of KLF10, Sei-1 is one of the target genes that may be regulated by Klf10 through promoter binding. The Sei-1 is a nuclear factor that implicated in cell cycle regulation through interaction with CDK4/CyclinD and E2F-1/DP-1 complexes. Ectopic expression of Sei-1 protein results in activation of the p21 gene and inhibition of cell growth via p53 mechanism; moreover, its knockout mice show a significant decrease in the amount of pancreatic β-islet. In this study, the ChIP-chip and ChIP-PCR were adopted to confirm whether Klf-10 directly binds to the Sp1/Klf binding sites on the upstream of Sei-1 promoter. Moreover, we also conducted RT-PCR, Western blotting, and promoter activity assay to investigate the regulation of Sei-1 by Klf10. Results demonstrated that Klf10 acts as a transcriptional activator on Sei-1 promoter where overexpression of Klf10 induces Sei-1 mRNA and protein expression in cells. After co-transfected with Klf10, Sei-1 promoter activity was significantly increased in which nearest binding site mutation showed loss of promoter activity. Results again suggested an important activating role of Klf10 on Sei-1 promoter. It has been known that Sei-1 is the key factor in cell cycle, it is unlikely to regulate p53 expression at the transcriptional level, and that it up-regulates the p53 activity probably by protein stabilization of p53, which in turn activates the p21 gene. Interestingly, Klf10 has been known as a dose-dependently activated p21 transcription that was independent of p53 and Sp1/Klf binding sites in p21 promoter. We attempted to confirm the presence of p21 using RT-PCR assay and Sei-1 shRNA in synchronized cells, which suggests that Klf10 activates p21 transcription indirectly through regulating the activity of Sei-1 promoter. In light of the influential effect of these two genes on pancreas, we compare the phenotype of pancreas between wild type and Klf10 knockout mice in the end of our study. Immunohistochemistry staining results indicated lower Sei-1 expression level and decreases in the number and amount of β-islet, as in the Sei-1 knockout mice, in our Klf10 knockout mice. Taken together, my experiments conclude that Klf10 up-regulates Sei-1 and thus affects related physiological functions.