MicroRNAs that target RGS5

Abstract

Objectives: An earlier meta-analysis on gene expression data derived from four microarray, two cDNA library, and one SAGE experiment had identified RGS5 as one of only ten non-housekeeping genes that were reported to be expressed in human trabecular meshwork (TM) cells by all studies. RGS5 encodes regulator of G-protein signaling-5. The TM tissue is the route of aqueous fluid outflow, and is relevant to the pathology of glaucoma. MicroRNAs constitute the most recently identified components of the cellular machinery for gene regulation in eukaryotic cells. Given our long standing interest in glaucoma, we aimed to identify miRNAs that may target RGS5.

Materials and methods: Eight miRNAs were selected for study using bioinformatics tools and available data on miRNAs expressed in the eye. Their effects were assessed using the dual luciferase assay. 3'-UTR segments of RGS5 mRNA were cloned downstream of a luciferase coding gene in psiCHECK2 vectors, and these were co-transfected with each of the miRNAs into HEK293 cells.

Results: The outcomes evidenced that one or more of the segments are in fact targeted by miR-7, miR-9, miR-96, miR-23a, miR-23b, miR-204, and miR-211. Down regulations by the miRNAs were statistically significant. The effect of miR-204 is considered particularly important as this miRNA is well known to regulate eye development and to affect multiple ocular functions.

Conclusion: Our results justify further studies on regulation of RGS5 expression and RGS5 downstream functions by these miRNAs.

Keywords: RGS5; miR-204; miR-211; miR-23a; miR-23b; miR-7; miR-9; miR-96.

Figure 1

Dual luciferase assay. A- Distribution of predicted target sites of miRNAs on 3’-UTR of GRS5. Nucleotide positions are with reference to the translation termination codon, and the position of the first nucleotide after that codon is considered +1. The thick lines represent RGS5-1, RGS5-2, and RGS5-3 that were cloned; the nucleotide positions at the termini of the fragments are shown. B- Dual luciferase assay results in HEK-293 cells. Standard deviations based on three replicative transfection experiments are shown. *indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001 for comparison of renilla/firefly luciferase ratios between control transfections and transfections with miRNAs. RLU, relative light units