Silencing of α-complex protein-2 reverses alcohol- and cytokine-induced fibrogenesis in hepatic stellate cells

Abstract

Background and aim: α-complex protein-2 (αCP2) encoded by the poly (rC) binding protein 2(PCBP2) gene is responsible for the accumulation of type I collagen in fibrotic livers. In this study, we silenced the PCBP2 gene using a small interfering RNA (siRNA) to reverse alcohol-and cytokine-induced profibrogenic effects on hepatic stellate cells (HSCs).

Methods: Primary rat HSCs and the HSC-T6 cell line were used as fibrogenic models to mimic the initiation and perpetuation stages of fibrogenesis, respectively. We previously found that a PCBP2 siRNA, which efficiently silences expression of αCP2, reduces the stability of type I collagen mRNA. We investigated the effects of the PCBP2 siRNA on cell proliferation and migration. Expression of type I collagen in HSCs was analyzed by quantitative real-time PCR and western blotting. In addition, we evaluated the effects of the PCBP2 siRNA on apoptosis and the cell cycle.

Results: PCBP2 siRNA reversed multiple alcohol- and cytokine-induced profibrogenic effects on primary rat HSCs and HSC-T6 cells. The PCBP2 siRNA also reversed alcohol- and cytokine-induced accumulation of type I collagen as well as cell proliferation and migration. Moreover, the combination of LY2109761, a transforming growth factor-β1 inhibitor, and the PCBP2 siRNA exerted a synergistic inhibitive effect on the accumulation of type I collagen in HSCs.

Conclusions: Silencing of PCBP2 using siRNA could be a potential therapeutic strategy for alcoholic liver fibrosis.

Keywords: Epidermal growth factor (EGF); Hepatic stellate cells; LY2a09761; Liver fibrosis; Migration; Myofibroblast; Platelet-derived growth factor (PDGF); Poly (rC) binding protein (PCBP)2; Primary HSC; Transforming growth factor (TGF)-β.

Fig. 1. PCBP2 siRNA reverses alcohol-induced expression of type I collagen in primary HSCs

Primary rat HSCs were transfected with 50 nmol PCBP2 siRNA or scrambled siRNA for 24 h and then treated with 100 mmol alcohol for 48 h. (A) Levels of PCBP2, type I collagen, and type I collagen primary mRNAs were quantified by real-time RT-PCR. (B) Protein expression of type I collagen was determined by western blotting. Results are presented as the mean ± SD (n=3). *P<0.05; **P<0.01.

Fig. 2. Effect of PCBP2 siRNA and alcohol on apoptosis of primary rat HSCs (A) and HSC-T6 cells (B)

HSCs were treated with 50 nmol siRNA for 24 h or 100 mmol alcohol for 48 h. The cells were stained with Annexin-V-FITC and propidium iodide, and then analyzed by flow cytometry. Results are presented as the mean ± SD (n=3). *P<0.05; **P<0.01.

Fig. 3. PCBP2 siRNA reverses cytokine-induced expression of type I collagen in primary rat HSCs (A, B) and HSC-T6 (C, D) cells

The cells were transfected with 50 nmol siRNA for 24 h and then treated with TGF-β1 or PDGF for 12 h. (A, C) mRNA levels of PCBP2 and type I collagen were quantified by real-time RT-PCR. (B, D) Protein expression of type I collagen was determined by western blotting. Results are presented as the mean ± SD (n=3). *P<0.05; **P<0.01.

Fig. 4. PCBP2 siRNA reverses PDGF-induced cell proliferation of primary rat HSCs (A, C) and HSC-T6 cells (B, D)

The cells were transfected with 50 nmol siRNA for 24 h and then incubated with PDGF for 12 h. (A, B) Cell proliferation was measured by MTT assays. (C, D) Cell cycle analysis was performed by flow cytometry. Results are presented as the mean ± SD (n=3). *P<0.05; **P<0.01.

Fig. 5. PCBP2 siRNA inhibits cytokine-induced migration of primary rat HSCs

After transfection with 50 nmol PCBP2 siRNA for 24 h, primary rat HSCs were seeded onto transwell chambers and incubated for 4 h to allow migration through the membrane. The migrated cells were stained, and images were obtained at 200× magnification. Six random microscopic fields were counted in each transwell. Cells treated with scrambled siRNA were used as the negative control. Results are presented as the mean ± SD (n=3). *P<0.05; **P<0.01.

Fig. 6. PCBP2 siRNA inhibits cytokine-induced migration of HSC-T6 cells

After transfection with 50 nmol PCBP2 siRNA for 24 h, HSC-T6 cells were seeded onto transwell chambers and incubated for 4 h to allow migration through the membrane. The migrated cells were stained, and images were obtained at 200× magnification. Six random microscopic fields were counted in each transwell. Cells treated with scrambled siRNA were used as the negative control. Results are presented as the mean ± SD (n=3). *P<0.05; **P<0.01.

Fig. 7. PCBP2 siRNA inhibits alcohol-induced migration of primary rat HSCs

Primary rat HSCs were transfected with 50 nmol PCBP2 siRNA for 24 h and then incubated with 100 mmol alcohol for 24 or 48 h. The cells were then seeded onto transwell chambers and incubated for 4 h to allow migration through the membrane. The migrated cells were stained, and images were obtained at 200× magnification. Six random microscopic fields were counted in each transwell. Cells treated with scrambled siRNA were used as the negative control. Results are presented as the mean ± SD (n=3). *P<0.05; **P<0.01.

Fig. 8. Combinational treatment with PCBP2 siRNA and LY2109761

(A) HSC-T6 cells were transfected with 50 nmol PCBP2 siRNA three times on days 1, 3, and 5. LY2109761 (10 µmol) was added on day 3, and alcohol (100 mmol) was added on day 6. The cells were harvested for western blot analysis of type I collagen on day 7. (B) Primary rat HSCs were transfected with 50 nmol PCBP2 siRNA three times on days 1, 3, and 5. LY2109761 (10 µmol) was added on day 3, and alcohol (100 mmol) was added on day 6. The cells were harvested for western blot analysis of type I collagen on day 7. (C) Primary rat HSCs were transfected with 25 or 50 nmol PCBP2 siRNA for 24 h, followed by incubation with 100 mmol alcohol for another 48 h. LY2109761 (10 µmol) was added to the medium during the siRNA transfection and alcohol incubation periods. Protein expression of type I collagen was evaluated by western blotting.