MiR-9a-5p regulates proliferation and migration of hepatic stellate cells under pressure through inhibition of Sirt1

Figure 4 MiR-9a-5p suppresses the proliferation, migration and activation of hepatic stellate cells by directly binding to Sirt1 3’-UTR region. A: Alignment of miR-9a-5p with the target sequence in the WT 3’-UTR of Sirt1 and the site-directed mutant 3’-UTR of Sirt1 (using Target Scan). The effect of co-transfection with pmir-Report, pmir-Report Sirt1 3’-UTR (wild-type or mutant) on luciferase activity was assessed in HEK293T cells. Student’s t-test was used for statistical analysis (n = 3; ^bP < 0.01 vs control); B and C: qRT-PCR and Western blot analyses of Sirt1 levels in hepatic stellate cells (HSCs) under the treatment of a miR-9a-5p mimic or a miR-9a-5p inhibitor and their corresponding negative controls (n = 3; ^aP < 0.05, ^bP < 0.01 vs control); D: HSCs transfected with Sirt1 or a negative control were exposed to increased pressure. Then, the cells were seeded in 96-well plates, cultured for 72 h and observed with a microplate reader at 0, 12, 24 and 72 h. ^aP < 0.05 vs control; E: The cell cycle analysis of each group was conducted by FACS; F: qRT-PCR was used to detect the expression of α-SMA and Col1 after up-regulation of Sirt1. Data are presented as the mean ± SD. ^bP < 0.01 vs control; G: Overexpression of miR-9a-5p and Sirt1 inhibits HSC migration by about 30 % assessed by Transwell assay. ^bP < 0.01 vs control. qRT-PCR: Real-time quantitative polymerase chain reaction; UTR: Untranslated region; HSC: Hepatic stellate cell.