MiR-22-3p and miR-29a-3p synergistically inhibit hepatic stellate cell activation by targeting AKT3

Abstract

Hepatic fibrosis (HF) is a worldwide health problem for which there is no medically effective drug treatment at present, and which is characterized by activation of hepatic stellate cells (HSCs) and excessive extracellular matrix (ECM) deposition. The HF model in cholestatic rats by ligating the common bile duct was induced and the differentially expressed miRNAs in the liver tissues were analyzed by microarray, which showed that miR-22-3p and miR-29a-3p were significantly downregulated in bile-duct ligation (BDL) rat liver compared with the sham control. The synergistic anti-HF activity and molecular mechanism of miR-22-3p and miR-29a-3p by targeting AKT serine/threonine kinase 3 (AKT3) in HSCs were explored. The expression levels of miR-22-3p and miR-29a-3p were downregulated in activated LX-2 and human primary normal hepatic fibroblasts (NFs), whereas AKT3 was found to be upregulated in BDL rat liver and activated LX-2 cells. The proliferation, colony-forming, and migration ability of LX-2 were inhibited synergistically by miR-22-3p and miR-29a-3p. In addition, cellular senescence was induced and the expressions of the LX-2 fibrosis markers COL1A1 and α-SMA were inhibited by miR-22-3p and miR-29a-3p synergistically. Subsequently, these two miRNAs binding to the 3'UTR of AKT3 mRNA was predicted and evidenced by the luciferase reporter assay. Furthermore, the proliferation, migration, colony-forming ability, and the expression levels of COL1A1 and α-SMA were promoted and cellular senescence was inhibited by AKT3 in LX-2 cells. Thus, miR-22-3p/miR-29a-3p/AKT3 regulates the activation of HSCs, providing a new avenue in the study and treatment of HF.

Keywords: AKT3; HSC activation; Hepatic fibrosis; LX-2; miR-22-3p; miR-29a-3p.

Figure 1.

Differential expression of genes in BDL rat liver. (a) Microarray analysis for miRNAs was used in sham operated or BDL rat liver tissue, n = 3. Hierarchical cluster analysis was performed for differential expression miRNAs. Black, no difference in expression; bright green, low expression; bright red, high expression. (b and c) KEGG and GO analyses of miR-22-3p and miR-29a-3p. (A color version of this figure is available in the online journal.)

Figure 2.

miR-22-3p and miR-29a-3p expressions in activated LX-2, activated NFs. (a to c) qRT–PCR and Western blotting were used to detect COL1A1 and α-SMA mRNA and protein expression in LX-2. (d) IF images of activated LX-2 stained for α-SMA (red, ×200 magnification). Scale bars = 50 μm. (e and f) These two miRNAs were tested by qRT–PCR in LX-2 and NFs. (A color version of this figure is available in the online journal.)

Figure 3.

Histological analysis of rat liver in sham operation group and BDL group (n = 7 per group). (a) The rat liver tissue sections were subjected to H&E (hematoxylin-eosin staining), Sirius Red staining, and Masson’s trichrome (100 ×, scale bars = 200 µm; 200 ×, scale bars = 100 µm). (b) Representative pictures of Sirius Red polarized light on rat liver tissue sections (100 ×, scale bars = 100 µm; Type I collagen: red or yellow, Type III collagen: green). (c) Immunohistochemical staining for α-SMA in the central venous and portal regions of rat liver tissue sections (100 ×, scale bars = 200 µm; 200 ×, scale bars = 100 µm). (d and e) Quantification of the Masson and Sirius Red positive area. (f) Quantitation of mean integrated optical density (IOD) of α-SMA positive stained tissues. (A color version of this figure is available in the online journal.)

Figure 4.

miR-22-3p and miR-29a-3p expressions in BDL rat liver. (a to c) The COL1A1, α-SMA, and AKT3 mRNA and protein expression in the liver tissues of sham and BDL rats were examined by qRT–PCR (a) and Western blotting (b and c) n = 7 per group. (d) The α-SMA and AKT3 expression were examined by co-IF (α-SMA: green, AKT3: red, nuclear: blue; 200 ×, scale bars = 50 μm). (e) These two miRNAs were examined by qRT–PCR in liver tissues from sham and BDL rats. (A color version of this figure is available in the online journal.)

Figure 5.

Overexpression of miR-22-3p and miR-29a-3p synergistically restrain LX-2 proliferation. (a) These two miRNAs expressions were detected by qRT–PCR in LX-2 transfected with their mimics. (b)The proliferation of LX-2 ability was detected by CCK-8 assay. (c) Colony-forming ability was tested. (A color version of this figure is available in the online journal.)

Figure 6.

Overexpression of miR-22-3p and miR-29a-3p synergistically induce cellular senescence. (a) The function of these two miRNAs mimics on cellular senescence was detected by SA-β-gal staining analysis. Scale bars = 200 μm. (b) The p16 and p21 expressions were examined by co-IF (p16: green, p21: red, nuclear: blue; 200 ×, scale bars = 50 μm). (A color version of this figure is available in the online journal.)

Figure 7.

miR-22-3p and miR-29a-3p overexpression synergistically inhibit LX-2 migration and activation. (a) The migration ability of each group of cells transfected with these two miRNAs mimics was tested by Transwell assays. Scale bars = 200 μm. (b and c) The migration ability was tested in each group of cells transfected with these two miRNAs mimics. Scale bars = 500 μm. (d to f) Expressions of COL1A1 and α-SMA mRNA and protein were measured by qRT–PCR (d) and Western blot (e and f) in LX-2 transfected with these two miRNAs mimics. (A color version of this figure is available in the online journal.)

Figure 8.

Synergistic mechanism of miR-22-3p and miR-29a-3p. (a to d) The expressions of these two miRNAs were measured by qRT–PCR.

Figure 9.

Bioinformatics screening for the common bona fide target of miR-22-3p and miR-29a-3p. (a) Common target of these two miRNAs in four databases. (b to e) KEGG (b), pathway enrichment (c), Gene Ontology (d), and STRING (e) analysis of the 24 candidate target genes. (f) PPI network of AKT3. (A color version of this figure is available in the online journal.)

Figure 10.

miR-22-3p and miR-29a-3p regulate AKT3 expression in LX-2. (a to c) AKT3 expression was examined in activated LX-2 by qRT–PCR (a) and Western blotting (b and c). (d) co-IF analysis of α-SMA and AKT3 (AKT3: red, α-SMA: green; nuclear: blue; 200 ×, scale bars = 50 μm). (e to g) AKT3 expression was examined in LX-2 transfected with these two miRNAs mimics by qRT–PCR (e), Western blotting (f and g). (h to j) AKT3 expression was examined in LX-2 transfected with these two miRNAs inhibitors by qRT–PCR (h), Western blotting (i and j). (A color version of this figure is available in the online journal.)

Figure 11.

The common bona fide target gene of miR-22-3p and miR-29a-3p is AKT3. The minimum free energy (MFE) of AKT3 and miR-22-3p (a) or miR-29a-3p (b) was predicted by RNAhybrid and the respective binding sites. (c) Evolutionary conservation of these two miRNAs assessed using the ECR browser. (d) These two miRNAs binding sites on AKT3. Four plasmids were used in the luciferase reporter assay. Including the putative target sites of these two miRNAs: Position 2004–2011 (+) Position 1667–1674 (+); deletion of putative miR-22-3p target site: Position 2004–2011 (–) Position 1667–1674 (+); deletion of putative miR-29a-3p target site: Position 2004–2011 (+) Position 1667–1674 (–); deletion of putative target sites of both these two miRNAs: Position 2004–2011 (–) Position 1667–1674 (–). (A color version of this figure is available in the online journal.)

Figure 12.

AKT3 promotes LX-2 proliferation. (a to c) AKT3 expression was tested by qRT–PCR (a) and Western blot (b and c) in LX-2 transfected with AKT3-siRNA. (d to f) AKT3 expression was tested by qRT–PCR (d) and Western blot (e and f) in LX-2 transfected with pcDNA3.1-AKT3. (g and h) The CCK-8 assay detected the proliferation ability of each group of cells after the transfection of AKT3-siRNA (g) or pcDNA 3.1-AKT3 (h). (i) The colony-forming ability was detected in each group of cells after the transfection of AKT3-siRNA or pcDNA 3.1-AKT3. (A color version of this figure is available in the online journal.)

Figure 13.

AKT3 inhibits LX-2 cellular senescence. (a) The function of AKT3-siRNA or pcDNA3.1-AKT3 on cellular senescence was detected by SA-β-gal staining analysis. Scale bars = 200 μm. (b) The p16 and p21 expressions were examined by co-IF (p16: green, p21: red, nuclear: blue; 200 ×, scale bars = 50 μm). (A color version of this figure is available in the online journal.)

Figure 14.

AKT3 promotes LX-2 migration and activation. (a and b) The migration ability of each group of cells transfected with AKT3-siRNA or pcDNA3.1-AKT3 was tested by Transwell assays (a; scale bars = 200 μm) and wound healing assays (b; scale bars = 500 μm). (c to e) COL1A1 and α-SMA expressions were examined by qRT–PCR (c) and Western blot (d and e) in LX-2 transfected with AKT3-siRNA. (f to h) COL1A1 and α-SMA expressions were examined by qRT–PCR (f) and Western blot (g and h) in LX-2 transfected with pcDNA 3.1-AKT3. (A color version of this figure is available in the online journal.)