Figure 3.

Overexpression of miR-223 attenuates TAZ and GLI2 expression in the liver and directly inhibits Gli2 in HSCs. (A-D) C57BL/6J mice were injected with CCl₄ for 6 weeks and were given intravenous injection of Ad-Gfp or Ad-miR-223 at the end of the 4^th week. (n=6/group). The liver tissues were collected at the 6^th week. (A-B) Representative images of TAZ staining and IHH staining are shown. Quantification of the TAZ⁺ and IHH⁺ area per field was performed and is shown. (C) RT-qPCR analyses of Gli2, Gli3 and Opn in liver tissues. (D) Representative images of GLI2 (red), α-SMA (green), and nuclei (blue) in the livers are shown. (E) Using a miRNA database (http://www.targetscan.org/), putative binding sites of miR-223 were predicted in the 3'-UTR of Gli2 mRNA in mice. The dashed line represents complementary base pairs between miR-223 and Gli2 3'-UTR. (F) Primary mouse HSCs were transfected with miR-223 mimics and nonspecific miRNA mimics (NS) for 24 hours. The mRNA levels of Gli2 and Gli3 were analyzed by RT-qPCR (G) Primary hepatocytes were transfected with Ad-miR-223 for 24 hours followed by H₂O₂ (500 µM, 5h) treatment. The expression of miR-223 in hepatocytes and hepatocyte-derived EVs was analyzed by RT-qPCR. Values represent means ± SEM. In panels A-D, n=6/group; Panels F-G are from three independent experiments. *P< 0.05.