Figure 1

ROCK1 is a target of microRNA-146a in neural cells.

(A) The effect of microRNA-146a (miR-146a) on ROCK1 expression was assessed in neural SH-SY5Y cells using a luciferase reporter system. A vector expressing miR-146a and a psiCHECK-2 vector containing either the full length ROCK1 3′ UTR or a truncated 3′ UTR lacking the miR-146a binding sites were co-transfected into neural SH-SY5Y cells. In control cells the vector expressing miR-146a was replaced with a scrambled miR-146a vector (scramble). Fluorescence was measured 48 hours after transfection. MiR-146a bound to the full length ROCK1 3′ UTR and inhibited its activity, but did not bind to the truncated ROCK1 3′ UTR. (B) Real-time PCR was used to detect the mRNA expression level of ROCK1 in neural SH-SY5Y cells. Overexpression of miR-146a or scramble did not affect the relative ROCK1 mRNA level compared to neural cells without vectors (negative). Relative expression of ROCK1 mRNA was calculated using the ΔΔCT method. (C) Western blot was used to measure ROCK1 protein translation in neural cells. Overexpression of miR-146a significantly decreased expression of endogenous ROCK1 protein in neural SH-SY5Y cells compared to negative or scramble treated neural cells. (D) Overexpression of miR-146a significantly reduced the relative ROCK1 protein level compared to negative or scramble treated neural cells. All of the data are expressed as means ± SD of at least three independent experiments.