Figure 2

Identification of mir-199a-5p as a hepatic ER stress-related miRNA. (a) The relative level of miR-199a-5p after TG, DCA, and DMSO treatments in human hepatocyte HL-7702/L02 cells. (b and c) The kinetics of the expression levels of miR-199a-5p (b), GRP78, ATF6, and IRE1α (c) in TG- and DCA-treated HL-7702/L02 cells. The relative expression levels of RNA were quantified by qRT-PCR and normalized to U6 or GAPDH. Data are shown as means±S.D. of three independent experiments. **P<0.01. (d) The miR-199a-5p-binding site predictions for GRP78, ATF6, and IRE1α mRNAs by Targetscan 6.2. The underlined letters indicate the mutant sites. (e) Target validation using luciferase reporters in HEK293 cells. The relative luciferase activities of 3′UTR reporters containing wild-type (WT) or mutant (Mut) transcripts were assayed 48 h after co-transfection with the indicated miRNAs or scrambled NC RNA (NC). (f) QRT-PCR and western blotting analysis of GRP78, ATF6, and IRE1α mRNA and protein expression in HL-7702/L02 cells transfected with miR-199a-5p minics or NC RNA. The relative expression levels of RNA and proteins were normalized to GAPDH. For proteins, data are shown as the rate normalized to GAPDH in each sample. (g) The endogenous miR-199a-5p in HL-7702/L02 cells suppressed the luciferase activities of 3′UTR reporters containing WT, Mut or control transcripts 48 h after transfection. Data are shown as means±S.D. of three independent experiments. **P<0.01