Figure 5

MiR-199a-5p differentially regulates the expression of DDR1. (A) HepG2 or (B) SNU-182 cells were transfected with 10 nM miR-199a-5p precursor or 100 nM DDR1-siRNA. Cell lysates were obtained 48 hours after transfection for immunoblot analysis of DDR1 protein expression. GAPDH protein levels were determined as a loading control. Cells transfected with control miRNA precursor or control siRNA were used as controls. Lane M indicates the molecular weight marker and lane numbers 1 to 4 represent control miRNA precursor, miR-199a-5p precursor, control siRNA, and DDR1-siRNA respectively. Arrow, DDR1 protein; stars, protein bands resulting from nonspecific binding to DDR1-antibody. (C) For mRNA stability assays, HepG2 cells were plated in 24-well plates and transfected with 10 nM of miR-199a-5p, miR-control or 100 nM si-DDR1 as a positive control. After transfection for 6 hours, cells were incubated with or without 4 μg/ml of actinomycin D for additional 36 hours. Total RNA was extracted from cells and DDR1 mRNA was quantified by qRT-PCR described before. The expression levels of DDR1 are presented as values normalized against 10⁶ copies of β-actin transcripts. Data are shown from 3 independent experiments each performed in triplicate as mean ± SD. *p < 0.05 vs. respective controls.