FIG 5.

miR-27a regulates Hmgcr expression by translational repression in cultured hepatocytes. AML12 cells were transfected with 500 ng of the miR-27a expression plasmid or pcDNA3.1 (as a control). After 12 h of transfection, they were incubated with actinomycin D (5 μg/ml) for different times. (A) Hmgcr mRNA levels were plotted relative to the 0-h time point as described in Materials and Methods (n = 3). (B) Endogenous Hmgcr mRNA half-life estimation in AML12 cells upon the ectopic overexpression of miR-27a. The mRNA half-life of Hmgcr was measured over 24 h in the presence of 5 μg/ml of actinomycin D in control cells (transfected with pcDNA3.1) and miR-27a-transfected AML12 cells. The Hmgcr mRNA half-lives are presented as means ± SEM of data from three independent experiments. (C and D) Effect of transcriptional attenuation on endogenous Hmgcr protein levels in miR-27a-overexpressing hepatocytes. AML12 cells were transfected with either pcDNA 3.1 or the miR-27a expression plasmid and incubated with actinomycin D for different time points. (C) Western blot analysis of total proteins was carried out to probe for Hmgcr and vinculin. The relative Hmgcr protein levels normalized to vinculin levels at different time points are also shown. The normalized Hmgcr levels as fold changes over the control for every time point of actinomycin D treatment are also indicated (n = 3). (D) Bar plot showing the normalized Hmgcr protein levels expressed as fold changes over the corresponding controls for different time points of actinomycin D treatment from three experiments. (E) Relative expression of Hmgcr 24 h after transfection of the miR-27a expression plasmid in AML12 cells was determined by qPCR using gene-specific primers (n = 3). Hmgcr expression was normalized to β-actin mRNA expression in the same sample. Statistical significance was determined by Student’s t test (unpaired, 2 tailed). *, P < 0.05 (compared to the control). (F) Pulse-chase analysis of HMGCR in HuH-7 cells transfected with the miR-27a expression plasmid. After 24 h of transfection, the cells were labeled with [³⁵S]Met for an hour, followed by a chase up to 120 min. The cells were then lysed, immunoprecipitated with the anti-HMGCR antibody, and analyzed by autoradiography. (G) Densitometric analysis of ³⁵S-labeled HMGCR levels in mock- and miR-27a-transfected lysates.