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  • In the current study we first isolated primary

    2019-06-26

    In the current study, we first isolated primary HSCs from rat liver and then treated the Terfenadine with alcohol. As shown in Fig. 1, the mRNA levels of PCBP2 and type I collagen were increased significantly. In addition, the type I collagen mRNA level was more significantly increased in primary HSCs than in HSC-T6 cells. We infer that the primary HSCs isolated from a healthy rat were quiescent and therefore more sensitive to the alcohol stimulation. We also demonstrated that treatment of primary HSCs with the PCBP2 siRNA significantly suppressed the alcohol-induced mRNA levels of PCBP2 and type I collagen as well as the protein level of type I collagen (Figs. 1 and 8). It is known that activated HSCs produce more type I collagen upon initiation of fibrogenesis and upregulate PDGF and TGF-β1 secretion in the fibrotic environment. In our study, as shown in Fig. 3A, type I collagen expression was increased by PDGF and TGF-β1 stimuli. Treatment with the PCBP2 siRNA significantly decreased the expression of type I collagen at mRNA and protein levels. αCP2, which is encoded by the PCBP2 gene, binds to the 3′ end of type I collagen mRNA and increases its stability, leading to the accumulation of a large amount of collagen in the fibrotic liver. Here, we showed that treatment of alcohol- and cytokine-stimulated primary rat HSCs with the PCBP2 siRNA significantly inhibited the collagen accumulation. PDGF is a cytokine with the strongest mitogenic stimulatory effects on HSC proliferation. PDGF and its receptors are highly expressed in cirrhotic liver. As Lin and Chen reported, primary rat HSCs stimulated with PDGF for 24 h show a dose-dependent increase in cell proliferation. Adachi et al. reported that PDGF activates NAD(P)H oxidase in HSCs, leading to the generation of reactive oxygen species, which further activates p38 mitogen-activated protein kinase (MAPK) and induces the proliferation of HSCs. Similarly, we found a significant proliferation effect on both primary HSCs and HSC-T6 cells exerted by PDGF stimulation (Fig. 4A and B). This result is also in accordance with a report showing that microRNA-214 (mir-214) suppresses glioma proliferation by targeting PCBP2. Mir-214 binds to the 3′-untranslated region of PCBP2 mRNA, which leads to the degradation of PCBP2 mRNA. As a result, mir-214 inhibits the proliferation and growth of glioma cells. In contrast, the restoration of PCBP2 dramatically reverses these tumor-suppressive effects. PCBP2 has been also reported to regulate astrocyte proliferation after spinal cord injury and facilitate the progression of esophageal squamous cell carcinoma by regulating cellular proliferation and apoptosis. In this study, HSC cells were transfected with PCBP2 siRNA and then treated with PDGF. The PCBP2 siRNA remarkably blocked the PDGF-stimulated proliferation of HSCs. HSC migration is one of the major features in liver fibrogenesis. In addition to alcohol, several cytokines were used in this study to mimic the fibrotic environment in the liver. As Yang et al. reported, the stimulation of HSCs with PDGF, TGF-β1, or EGF enhanced the migratory capacity of HSCs. Furthermore, TGF-β1 has been reported to facilitate the migration of HSCs in a modified Boyden Chamber model via both chemotactic and haptotactic mechanisms. In addition, PDGF promotes the migration of murine HSC cell line GRX. Alcohol has also been reported to promote migration and invasion of triple-negative breast cancer cells through activation of p38 MAPK and c-Jun N-terminal kinase. In this study, PDGF, TGF-β1, EGF, and alcohol were used to exert a migration effect on HSCs. We found that treatment with PCBP2 siRNA attenuated or reversed the HSC migration induced by alcohol, PDGF, TGF-β1, and EGF, which is consistent with the study of Lin et al. who found that knocking down PCBP2 inhibits glioma cell migration and invasion via Rho GDP dissociation inhibitor alpha.