Research Article

Glycyrrhetinic Acid-Induced MiR-663a Alleviates Hepatic Stellate Cell Activation by Attenuating the TGF-β/Smad Signaling Pathway

Figure 2

Identification of TGF-β1 as a direct target gene of miR-663a. (a) The relative miR-663a expression in LX2 cells was measured by RT-qPCR. LX2 cells were transfected with miR-663a mimics and miRNA mimic negative control (miR-NC) for 72 h. U6 was used as an internal control. (b) The levels of TGF-β1, TGF-βRI, CD209, GLG1, INPPL1, IL4R, KLF10, STAM, TLN1, USF2, and UBA52 were measured by RT-qPCR after LX2 cells were transfected with miR-663a mimics and miRNA mimic negative control (miR-NC) for 72 h. GAPDH was used as an internal control. The results are expressed as the mean; error bars denote the standard error of the mean. , compared with the miR-NC group. (c) The three potential seed sequences of miR-663a in the 3′-UTR of TGF-β1 are indicated. Wild-type (WT) TGF-β1: the putative binding site of miR-663a on the 3′UTR of TGF-β1 predicted by TargetScan software. Mutant (MUT) TGF-β1: the mutant sequences of miR-663a seed region on the 3′-UTR of TGF-β1. (d) Dual-luciferase reporter assay was performed to validate the direct binding between TGF-β1 3′-UTR and miR-663a. After subcloning the WT or MUT of TGF-β1 3′-UTR into a luciferase reporter vector, we cotransfected the miR-663 mimics or miRNA mimic negative control (miR-NC) with the vector inserted with WT or MUT TGF-β1 3′-UTR into the LX2 cells. Then, the luciferase activity was examined. , compared with the WT + miR-NC group.
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