Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
MicroRNA-453 Modulates Sonic Hedgehog Gene Expression in Human Chondrocytes by Directly Targeting Its Open Reading Frame.
Akhtar1, Nahid, Haqqi2, Tariq M.
MicoRNAs (miRNAs) are small endogenous, non-coding RNAs that are important post-transcriptional regulators of gene expression. Sonic Hedgehog (SHH) morphogen is an essential signaling molecule required for numerous processes during development and has recently been implicated in OA pathogenesis. However, regulation of SHH expression in OA remains unclear. Here we studied (a) whether IL-1b-induce the expression of SHH; and (b) whether miRNAs play a role in regulating expression of SHH in human chondrocytes.
Chondrocytes were derived by enzymatic digestion of human cartilage from OA patients (OA chondrocytes) undergoing total knee arthroplasty. Chondrocytes were stimulated with IL-1b (5ng/ml) in vitro and total RNA was prepared. Expression of SHH and its downstream targets PTCH-1, GLI-1, HHIP, ADAMTS-5 and MMP-13 was quantified by TaqMan Assays. miRGen Targets (http://diana.pcbi.upenn.edu/cgi-bin/miRGen/v3/Targets.cgi) was used to identify miRNAs that potentially target the SHH mRNA and the free energy scores of selected miRNA:mRNA hybrids were determined using RNAHybrid program (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid). miRNAs were purified using the mirVANA system and single stranded cDNA was synthesized with the stem loop-specific primers and the expression of miRNAs was quantified using TaqMan Assays. HEK293 cells were co-transfected with pre-miRNAs, antagomirs and SHH expression plasmid and the protein expression was determined by Western Immunoblotting. Data was analyzed using Origin 6.1 software package and p<0.05 was considered significant.
Our results showed that the expression of of SHH was higher in damaged cartilage (stained with India ink, slight or no loss of Safranin O staining) compared to smooth cartilage (no staining with India ink, little or no loss of Safranin O staining) obtained from OA patients at the time of knee arthrthroplasty. The expression of SHH signaling genes and their downstream targets (PTCH-1, GLI-1, and HHIP, MMP-13, ADAMTS-5, COL10A1 and RUNX-2) was also higher in damaged cartilage. A full length search of SHH transcript identified several miRNAs including Hsa-miR-453, Hsa-miR-602 and Hsa-miR-608 with seed matched sites within the coding sequence of SHH mRNA. IL-1b-stimulation (10 ng/ml) resulted in significant up-regulation of SHH mRNA (~11.3-fold at 6h; ~15.5-fold at 12 h) and protein levels in chondrocytes obtained from damaged cartilage. In addition, IL-1b-stimulation also down regulated the expression of Hsa-miR-453 (~4.6-fold at 6h; ~11.6-fold at 12 h), Hsa-miR-602 (~3.9-fold at 6h; ~2.9-fold at 12 h) and Hsa-miR-608 (~3.6-fold at 6h; ~7.5-fold at 12 h) in these chondrocytes. Co-transfection of HEK-293 cells with the SHH expression plasmid and the pre-miR-453 resulted in significant inhibition of SHH mRNA and protein expression, while the transfection of SHH expression plasmid with the antagomirs had no effect on the SHH mRNA and protein expression.
Our findings indicate that miR-453 regulates the expression of SHH in OA chondrocytes by directly targeting its coding sequence. This provides an important and novel perspective for developing therapeutic strategies to treat OA.
To cite this abstract, please use the following information:
Akhtar, Nahid, Haqqi, Tariq M.; MicroRNA-453 Modulates Sonic Hedgehog Gene Expression in Human Chondrocytes by Directly Targeting Its Open Reading Frame. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :2437