MicroRNA-602 and MicroRNA-608 Regulate Sonic Hedgehog Expression via Target Sites in the Coding Region in Human Chondrocytes

Objective. Hedgehog (HH) signaling has recently been associated with cartilage degradation in osteoarthritis (OA). Because interleukin-1 beta (IL-1 beta) has been implicated as a principal instigator of OA, we sought to determine whether IL-1 beta induces the expression of sonic HH (SHH) and its regulation by microRNAs (miRNAs) in human chondrocytes. Methods. Expression of SHH protein in human OA cartilage and in an animal model of OA was determined by immunohistochemical analysis and immunofluorescence analysis, respectively. Gene and protein expression in IL-1 beta- or SHH-stimulated OA chondrocytes was determined by TaqMan assays and Western blotting, respectively. The effect of overexpression of miRNA-602 (miR-602) and miR-608 or their antagomirs on SHH expression was evaluated by transient transfection of human chondrocytes and HEK 293 cells. The role of signaling pathways was evaluated using small molecule inhibitors. Binding of miRNAs with the putative seed sequence in SHH messenger RNA (mRNA) was validated using a luciferase reporter assay. Results. Expression of SHH, patched 1, Gli-1, HH-interacting protein, matrix metalloproteinase 13 (MMP-13), and Col alpha 1(X) was high in damaged OA cartilage. In damaged cartilage and in IL-1 beta-stimulated OA chondrocytes, expression of SHH was inversely correlated with expression of miR-608. Cotransfection of OA chondrocytes with miR-608 or miR-602 mimic inhibited reporter activity, and mutation of the miRNA seed sequences abolished the repression of reporter activity. Overexpression of miR-602 or miR-608 inhibited the expression of SHH mRNA and protein, and this was abrogated by antagomirs. Stimulation with recombinant human SHH protein upregulated MMP-13 expression, and inhibition of HH signaling blocked MMP-13 expression in OA chondrocytes. Conclusion. MiR-602 and miR-608 are important posttranscription regulators of SHH expression in OA chondrocytes, and their suppression by IL-1 beta may contribute to the enhanced expression of SHH and MMP-13 in OA.