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Tomohiro Masuda, Anitha Yerrabelli, Donald J. Zack; Exploration Of The Target Gene Independent Mechanism By Which Shrna Regulates Rhodopsin Promoter Activity. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5135.
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To better understand the target independent mechanism by which shRNAs can modulate the activity of rhodopsin-promoter and other reporter constructs.
Bovine rhodopsin promoter reporters were transfected into HEK293, COS7, and WERI retinoblastoma cells with or without CRX, NRL, and shRNA expression vectors. The reporters used were firefly (Fluc) and Gaussia (Gluc) luciferase and mRFP. The shRNAs used targeted EGFP and Pias2 genes. Various shRNAs with random and Pias2-based mutation stem structure were also used. The culture medium or the cells were collected at designated time points to measure luciferase activity. mRFP signal was detected and analyzed using a Cellomics image-analysis system. Cells were also collected and analyzed for endogenous gene and miRNA expression by quantitative real-time PCR (qPCR), microarray, and nCounter miRNA expression assay.
In HEK293 cells, CRX and NRL induced Gluc activity ~7 fold compared to control. Surprisingly, reporter activity was enhanced synergistically to a maximum of ~100 fold in the presence of several shRNAs, including control shRNAs. However, shRNA alone, without CRX and NRL, induced the Gluc activity ~5 fold at most. Some shRNAs, however, had little or no induction of the Gluc activity both by itself and in the presence of CRX and NRL. Transfection of mutant shRNAs identified a critical nucleotide for the activity from the stem of the shRNA - replacing the key nucleotide largely eliminated the ability of the shRNA to enhance CRX- and NRL-mediated Gluc activity. Results were similar with the other reporters and cell lines. Microarray and qPCR analysis did not identify any gene expression changes that could explain the observed shRNA-mediated reporter effects. miRNA expression studies to explore whether the observed shRNA effects could be mediated by modulation of miRNA activity are underway.
Our results indicate that the synergistic enhancement of rhodopsin promoter activity by shRNAs in the presence of CRX and NRL is target gene-independent but shRNA sequence-specific. We have identified a critical nucleotide in the stem of the shRNA that appears crucial for this enhancing activity. Further research is required to elucidate the mechanism of this unique shRNA function.
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