P 0. 05 were considered statistically significant. Results IL 1b treatment increases expression of miR 146a and VEGF and decreases Smad4 expression in chondrocytes To identify the miRNAs involved in pathogenesis of OA, we screened for miRNAs responsive to treatment of Vandetanib mechanism of action the proinflammatory cytokine IL 1b in primary rat chondrocytes. This is an established cell culture model to mimic inflammation and other molecular events related to the progression of OA in chondrocytes. Expression of miRNAs in IL 1b stimulated chon drocytes was investigated by microarray analysis. A series of miRNAs changed their expression levels in response to IL 1b treatment. Of particular interest, miR 146a was chosen for further investigation because previous studies have revealed that miR 146a mediates inflamma tion response, and its expression is higher in OA cartilage than in normal cartilage.
Treatment of IL 1b rapidly induced miR 146a within 6 hours in primary rat chondrocytes, and its expression gradually increased over a 24 hour time course, which is consistent with the microarray results. In parallel with the increase of miR 146a level, IL 1b treat ment stimulated VEGF mRNA and protein levels Inhibitors,Modulators,Libraries in a time dependent manner. In con trast, IL 1b treatment inhibited Smad4 mRNA and protein levels in a time dependent manner. miR 146a directly inhibits Smad4 expression through a seed site in the 3 UTR of Smad4 mRNA To determine whether miR 146a regulates the expres sion of Smad4 and VEGF, we transfected miR 146a into primary chondrocytes. Overexpression of miR 146a inhibited Smad4 protein levels and stimulated VEGF protein levels.
Conversely, Inhibitors,Modulators,Libraries transfection of a miR 146a inhibitor stimulated Smad4 protein levels and inhibited VEGF protein levels in chondrocytes. miR 146a thus regulates the expression of Smad4 and VEGF in an opposite manner. Using miRNA target prediction software, we iden tified a potential miR 146a binding sequence in the 3 UTR of Smad4. To determine whether miR 146a inhibits Smad4 expression through this seed sequence, we constructed luciferase reporter plasmids harboring the wildtype 3 UTR and the mutant 3 UTR in which the putative miR 146a binding site is mutated. While the reporter activity of the wildtype 3 UTR is significantly inhibited by miR 146a, this inhi bition is greatly reduced in the mutant 3 UTR. Smad4 is thus a direct target of miR 146a.
IL 1b regulates Smad4 and VEGF expression through miR 146a To elucidate the role of miR 146a in mediating IL 1b signaling, we used Inhibitors,Modulators,Libraries a specific miR 146a hairpin inhibitor to block its expression. Chondrocytes were treated with IL 1b for Inhibitors,Modulators,Libraries 24 hours in the presence or absence of the miR 146a inhibitor. Knockdown of endogenous Inhibitors,Modulators,Libraries miR 146a with the inhibitor significantly suppressed selleck screening library the IL 1b upregulation of miR 146a expression.