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Y. Zhang, R. Converse, W.-Y. Kao, C.-Y. Liu; Pax6 Up-Regulates p21 Expression in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4291.
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© ARVO (1962-2015); The Authors (2016-present)
Pax6, a pair- and homeo-box- transcription factor, plays a key role in eye development. Our previous results indicated that overexpression of Pax6 suppresses cell proliferation and retards cell cycling in a rabbit corneal epithelial cell line (SIRC). This study initially characterizes the mechanism by which Pax6 may regulate the expression of CDK inhibitors in SIRC cells.
A SP-10 stable cell line, SIRC cell carrying a full length Doxcycline (DOX) inducible Pax6 transgene, was cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 500ug/ml G418. Total RNA was extracted with TriZol reagent from DOX induced SP-10 cells, using non-induced SP-10 cells as a control. 5ug total RNA was reverse transcribed with the ImProm-IITM Reverse Transcription kit (Promega, Madison, WI) according to manufacture’s instruction. Real-time PCR was performed to determine the expression levels of p21, p27, p57, and Rb against GAPDH using Q-Gene software in conjunction with a real-time PCR system. Pax6 binding sites in p21, p27, p57, and Rb were searched via bioinformatic analysis.
Real-time PCR showed that the mRNA expression level of p21 increased by excess Pax6 within 6 hours and reached a 7-fold increases at 24 hours as compared to non-induced SP-10 cells. While the mRNA expression levels of p27, p57 and Rb did not change after DOX induction. Bioinformatic analysis revealed that several potential Pax6-binding sites were found in the rabbit non-coding genomic DNA sequence of p21, three of which located upstream of the p21 transcription start site and three located in p21 intron one. No Pax6 binding elements were found in the rabbit p27, p57, Rb genomic DNA sequence, consistent with the finding of mRNA expression levels determined by real-time PCR.
These data implicated that Pax6 may regulate cell cycling through increasing or activating p21 gene expression but not other CDK inhibitors in this cell line, e. g. p27, p57.
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