The transcriptional activity of WT1 gene promoter and enhancer in cell lines with diverse tissue origin.

摘要

In this study, tissue-specific transcriptional activity of WT1 promoter and enhancer was investigated in a hope to design gene therapy strategy based on the transcriptional regulatory elements of WT1 gene. WT1 promoter and/or enhancer were ligated into pEGFP-1 to construct recombinant vectors with EGFP gene as a reporter and analyzed using Flow cytometry. The results showed that pEWP containingWT1 promoter induced the highest EGFP expression in ECV304 at 16.54 +/- 2.45 times, mildly higher in MCF-7 and SHG44 at 9.46 +/- 1.10 and 7.29 +/- 0.73 times, and in K562 cell line at 2.93 +/- 0.27 times, compared to that of pEGFP-1. However, pEWPA, with WT1 enhancer inserted at Afl II site, increased basal transcription levels of the WT1 promoter in HT-29, SHI-1 and K562 cells by 4.81, 3.06 and 1.01-fold, respectively. pEWPD inserted at NotI and pEWPE inserted at BamI had no ability to increase the transcriptional activity of WT1 promoter. Moreover, reporter gene silenced was observed in transfected host cells by flow cytometryt and real-time PCR. These results suggested that transcriptional activities of WT1 promoter in the recombinant vector seemed not correlated to the constitutional expression level of endogenous WT1 gene. The WT1 enhancer could promote the transcriptional activities of WT1 promoter in some of the cell lines regardless of the hematopoietic tissue origin. The inserted site of enhancer in vector influenced the transcriptional activity of promoter and the extent of reporter gene silencing exerted the influence on the analysis of transcriptional activity.