Overview of miRNA repression

miRNA are short (22-24 nt) strands of RNA known to regulate gene expression through repression of mRNA. mRNA that contain the complementary sequence to the miRNA are targeted by the RNA-induced silencing complex and are selectively degraded, thus repressing protein production.

Our goal is to use miRNA as a signal for cell-cell communication. We believe this can be accomplished by the packaging of miRNA into exosomes, which would then carry the miRNA signal to a receiver cell. Further, certain miRNA seem to be selectively targeted to exosomes, through a mechanism which is poorly understood. It has been shown that in Jurkat T cells, exosomes are naturally enriched in miR-451 and miR-503. For this reason, we chose to use Jurkat cells as our sender cells and to design a receiver circuit which could detect these two miRNA. For our receiver cells, we have chosen HEK293 cells because they are well characterized, easy to culture, and easy to transfect.

eYFP-target Characterization

The simplest receiver circuit is composed of constitutively expressed eYFP (under the hEF1a promoter), designed with target sites for either miR-451 or miR-503 in the 3' UTR. In addition, we include constitutively expressed tagBFP (also under the hEF1a promoter) as a control for transfection efficiency. This allows us to distinguish cells showing repression from cells that were simply not transfected efficiently.

To test the receiver circuit, we designed and had synthesized siRNA corresponding to miR-451 and miR-503. Co-transfection of these siRNA and the receiver circuit in the same cells allows us to characterize the sensitivity of the eYFP reporter and demonstrate that we can detect the silencing affect, and gives us confidence that the reporter will be sensitive to the natural miRNAs as well.

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Exosome Isolation and Co-Culturing