mir-142-3p regulates the Bmal1 mRNA and protein levels. (A) Total cell RNA and protein were extracted from the six cell lines (NIH3T3, 293ET, MCF-7, U87MG, T98G and U251), and the expression of mir-142-3p as well as the BMAL1 protein were determined by quantitative RT-PCR (mean ± SD, n = 3) and western blotting, respectively. (B-C) Ectopic expression of mir-142-3p in NIH3T3 and 293ET cells inhibited the expression of Bmal1/BMAL1. Due to the low transfection efficiency of plasmids, the NIH3T3 cells were transfected with synthetic mir-142-3p mimics (mir-142-3p) or control mimics (mir-NC) (B). The 293ET cells were transfected with the mir-142 expression plasmid (mir-142-pcDNA3.1) or control vector (pcDNA3.1) (C). The over-expression of mir-142-3p in NIH3T3 and 293ET cells was first verified, and then the Bmal1/BMAL1 mRNA and protein levels were detected by quantitative RT-PCR (mean ± SD, n = 3) and western blotting, respectively. The expression levels of mir-142-3p in control NIH3T3 (mir-NC) and 293ET (pcDNA3.1) cells were normalized against that in mir-142-3p/mir-142-pcDNA3.1-transfected cells which were set at 1. Note that, because the mir142-3p expression was low in control 293ET cells (pcDNA3.1) and was increased more than 1000-fold when cells were transfected with mir-142-pcDNA3.1, the relative expression level of mir-142-3p in control group (pcDNA3.1) was very low. (D) The endogenous mir-142-3p in U87MG cells was knocked down by transfection with synthetic mir-142-3p antagomirs (anti-142-3p). The expression level of mir-142-3p and BMAL1 mRNA in U87MG cells were determined by quantitative RT-PCR (mean ± SD, n = 3) and normalized against controls set at 1. All of the western blotting results were quantified from the pixel values in grayscales (mean ± SD, n = 3) and normalized against controls set at 1. Inserts are the representative western blotting results. *P < 0.05 and **P < 0.01.