Salvianolic acid B-induced microRNA-152 inhibits liver fibrosis by attenuating DNMT1-mediated Patched1 methylation - PubMed (original) (raw)

Salvianolic acid B-induced microRNA-152 inhibits liver fibrosis by attenuating DNMT1-mediated Patched1 methylation

Fujun Yu et al. J Cell Mol Med. 2015 Nov.

Abstract

Epithelial-mesenchymal transition (EMT) was reported to be involved in the activation of hepatic stellate cells (HSCs), contributing to the development of liver fibrosis. Epithelial-mesenchymal transition can be promoted by the Hedgehog (Hh) pathway. Patched1 (PTCH1), a negative regulatory factor of the Hh signalling pathway, was down-regulated during liver fibrosis and associated with its hypermethylation status. MicroRNAs (miRNAs) are reported to play a critical role in the control of various HSCs functions. However, miRNA-mediated epigenetic regulations in EMT during liver fibrosis are seldom studied. In this study, Salvianolic acid B (Sal B) suppressed the activation of HSCs in CCl4 -treated mice and mouse primary HSCs, leading to inhibition of cell proliferation, type I collagen and alpha-smooth muscle actin. We demonstrated that the antifibrotic effects caused by Sal B were, at least in part, via inhibition of EMT and the Hh pathway. In particular, up-regulation of PTCH1 was associated with decreased DNA methylation level after Sal B treatment. Accordingly, DNA methyltransferase 1 (DNMT1) was attenuated by Sal B in vivo and in vitro. The knockdown of DNMT1 in Sal B-treated HSCs enhanced PTCH1 expression and its demethylation level. Interestingly, increased miR-152 in Sal B-treated cells was responsible for the hypomethylation of PTCH1 by Sal B. As confirmed by the luciferase activity assay, DNMT1 was a direct target of miR-152. Further studies showed that the miR-152 inhibitor reversed Sal B-mediated PTCH1 up-regulation and DNMT1 down-regulation. Collectively, miR-152 induced by Sal B, contributed to DNMT1 down-regulation and epigenetically regulated PTCH1, resulting in the inhibition of EMT in liver fibrosis.

Keywords: DNA methylation; DNA methyltransferase; Patched1; hepatic stellate cells; microRNA-152.

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Figures

Figure 1

Effects of Sal B on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. (A) Hematoxylin and eosin staining (×100) and Masson staining (×100) for assessing liver fibrosis. (B) The level of alpha-smooth muscle actin (α-SMA) was analysed by immunohistochemistry in CCl4 mice after Sal B treatment. Representative views from each group are presented (original magnification, ×10). The mRNA levels of α-SMA (C) and Col1A1 (E) were detected in CCl4 mice after Sal B treatment. The protein levels of α-SMA (D) and type I collagen (F) were detected in CCl4 mice after Sal B treatment. GAPDH was used as internal control. *P < 0.05 compared to the control and #P < 0.05 compared to the model. Each value is the mean ± SD of three experiments.

Figure 2

Effects of Sal B in mouse primary hepatic stellate cells (HSCs). (A) Phase-contrast microscopy. HSCs were isolated from mouse liver (Day 1) and cultured for the indicated periods. Scale bar 100 μm. Black arrows indicated quiescent HSC phenotype and activated HSC phenotype, respectively, in Day 1 and Day 4 (7). (B) The mRNA levels of alpha-smooth muscle actin (α-SMA) and Col1A1 were analysed by real-time PCR. *P < 0.05 compared to Day 1. (C) The protein levels of α-SMA and type I collagen were analysed by Western blot. GAPDH was used as internal control. *P < 0.05 compared to Day 1. (D) The mRNA levels of α-SMA and Col1A1 were inhibited by Sal B, which were reversed by Patched1 (PTCH1) siRNA. *P < 0.05 compared to Day 4 and #P < 0.05 compared to Sal B group. The primary HSCs were studied 4 days after isolation and treated with Sal B for 48 hrs. (E) The protein levels of α-SMA and type I collagen were reduced by Sal B, which were reversed by PTCH1 siRNA. GAPDH was used as internal control. *P < 0.05 compared to Day 4 and #P < 0.05 compared to Sal B group. (F) Cell proliferation was determined by the MTT assay. (G) Cell apoptosis was determined by Caspase3 activity assay. The primary HSCs were studied 4 days after isolation. Primary HSCs were transfected with PTCH1 siRNA, DNA methyltransferase 1 (DNMT1) siRNA or miR-152 inhibitor for 48 hrs and treated with Sal B for an additional 48 hrs. *P < 0.05 compared to the control and #P < 0.05 compared to Sal B group. Each value is the mean ± SD of three experiments.

Figure 3

Effects of Sal B on epithelial-mesenchymal transition (EMT) in vitro and in vivo. The primary hepatic stellate cells (HSCs) were studied 4 days after isolation and treated with Sal B for 48 hrs. (A) Immunofluorescence staining for desmin (green), vimentin (red) and E-cadherin (red) were evaluated by confocal laser microscopy. 4,6-diamidino-2-phenylindole (DAPI) stained nuclei blue. Scale bar, 50 μm. (B) The mRNA levels of desmin, vimentin and E-cadherin were reduced by Sal B. *P < 0.05 compared to the control. (C) The protein levels of desmin, vimentin and E-cadherin were inhibited by Sal B. GAPDH was used as internal control. *P < 0.05 compared to the control. (D) The mRNA levels of desmin, vimentin and E-cadherin in carbon tetrachloride (CCl4) mice were reduced after Sal B treatment. *P < 0.05 compared to the model. (E) The protein levels of desmin, vimentin and E-cadherin in CCl4 mice were inhibited after Sal B treatment. GAPDH was used as internal control. *P < 0.05 compared to the model. Each value is the mean ± SD of three experiments.

Figure 4

Sal B contributed to the suppression of cell migration in hepatic stellate cells (HSCs). The primary HSCs were studied 4 days after isolation and treated with Sal B for 48 hrs. (A) Effects of Sal B on cell migration were tested by wound healing. Dashed line indicates edge of cell migration. (B) Effects of Sal B on cell migration were tested by transwell migration assay. Five fields of migrated cells in the lower side of transwell were counted with a microscope at ×100. *P < 0.05 compared to the control. Each value is the mean ± SD of three experiments.

Figure 5

Effects of Sal B on Hh signalling pathway in vitro and in vivo. The primary hepatic stellate cells (HSCs) were studied 4 days after isolation. Primary HSCs were transfected with DNA methyltransferase 1 (DNMT1) siRNA for 48 hrs and treated with Sal B for an additional 48 hrs. (A) The mRNA levels of Patched1 (PTCH1), Smo and Gli2 were analysed by real-time PCR. *P < 0.05 compared to the control. #P < 0.05 compared to Sal B group. (B) The protein levels of PTCH1, Smo and Gli2 were analysed by western blot. GAPDH was used as internal control. *P < 0.05 compared to the control and #P < 0.05 compared to Sal B group. (C) The mRNA levels of PTCH1, Smo and Gli2 were analysed by real-time PCR in mice. *P < 0.05 compared to the control and #P < 0.05 compared to the model. (D) The protein levels of PTCH1, Smo and Gli2 were analysed by western blot in mice. GAPDH was used as internal control. *P < 0.05 compared to the control and #P < 0.05 compared to the model. Each value is the mean ± SD of three experiments.

Figure 6

Patched1 (PTCH1) expression was regulated by promoter DNA methylation. The primary hepatic stellate cells (HSCs) were studied 4 days after isolation. Primary HSCs were transfected with DNA methyltransferase 1 (DNMT1) siRNA for 48 hrs and treated with Sal B for an additional 48 hrs. (A) A schematic representation of the promoter region amplified by bisulfide sequencing and ChIP-PCR assay. F1 represents region selected for bisulphite sequencing. Each vertical bar represents the presence of a CpG dinucleotide. F2 represents region selected for chromatin immunoprecipitation analysis. Promoter DNA methylation of PTCH1 was detected by bisulfide sequencing in mice (B) and primary HSCs (C). The average percentage of DNA methylation was shown at the end of each row.

Figure 7

Patched1 (PTCH1) promoter methylation is associated with the activity of DNA methyltransferase 1 (DNMT1). The primary hepatic stellate cells (HSCs) were studied 4 days after isolation and treated with Sal B for 48 hrs. (A) The mRNA levels of DNMT1, DNMT3a and DNMT3b were analysed by real-time PCR in primary HSCs. *P < 0.05 compared to the control. (B) DNMT1, DNMT3a and DNMT3b were analysed by western blot. GAPDH was used as an internal control. *P < 0.05 compared to the control. (C) The mRNA levels of DNMT1, DNMT3a and DNMT3b were analysed by real-time PCR in carbon tetrachloride (CCl4)-treated mice after Sal B treatment. *P < 0.05 compared to the model. (D) DNMT1, DNMT3a and DNMT3b were analysed by western blot in CCl4-treated mice after Sal B treatment. GAPDH was used as an internal control. *P < 0.05 compared to the model. (E) The bindings of MeCP2 and DNMT1 to the promoter region of PTCH1 in HSCs transfected with DNMT1 siRNA. (F) The bindings of MeCP2 and DNMT1 to the promoter region of PTCH1 in HSCs transfected with miR-152 inhibitor. Quantitative-chromatin immunoprecipitation assay was performed to detect the binding of MeCP2 or DNMT1 to the CpGs of PTCH1 using specific antibodies against MeCP2 or DNMT1. The data were expressed as mean ± SD, representing the relative levels of amplification at region F2 after chromatin immune precipitation with specific antibodies against MeCP2 or DNMT1 with normalization by total input DNA.

Figure 8

miR-152 was up-regulated by Sal B and suppresses DNA methyltransferase 1 (DNMT1) expression by targeting the 3′-untranslated region (3′-UTR) sequences of DNMT1. (A) The relative expression levels of miR-152, miR-148a and miR-148b were analysed in Sal B-treated hepatic stellate cells (HSCs) by real-time PCR. The primary HSCs were studied 4 days after isolation and treated with Sal B for 48 hrs. *P < 0.05 compared with the control. (B) The relative expression levels of miR-152, miR-148a and miR-148b were analysed in carbon tetrachloride (CCl4)-induced mice after Sal B treatment by real-time PCR. *P < 0.05 compared with the control. (C) Putative miR-152 binding sites (TS) within the mouse DNMT1 3′-UTR are shown. The position of the binding sites was numbered relative to the first nucleotide of the 3′-UTR. Mutations were introduced into DNMT1 3′-UTR that matched the seed region of miR-152 as shown in DNMT1 Mu. (D) Dual-luciferase assay were performed in 293T and mouse primary HSCs cotransfected with the firefly luciferase constructs containing the DNMT1 wild-type or Mu 3′-UTR and miR-152 mimics or scrambled oligonucleotides as the negative control. Each value is the mean ± SD of three experiments.

Figure 9

Overexpression of miR-152 inhibited DNA methyltransferase 1 (DNMT1) expression and reduced the methylation of Patched1 (PTCH1), leading to the restoration of PTCH1 expression. Primary hepatic stellate cells (HSCs) were transfected with miR-152 inhibitor for 48 hrs and treated with Sal B for an additional 48 hrs. (A) Promoter DNA methylation of PTCH1 was detected by bisulfide sequencing. The average percentage of DNA methylation was shown at the end of each row. (B) The mRNA levels of DNMT1 and PTCH1 were analysed by real-time PCR. (C) The protein levels of DNMT1 and PTCH1 were analysed by western blot. GAPDH was used as internal control. *P < 0.05 compared with the control and #P < 0.05 compared with Sal B group. Each value is the mean ± SD of three experiments.

Figure 10

The signalling pathway was discovered in hepatic stellate cells (HSCs) after Sal B treatment. Sal B induces miR-152 up-regulation, DNA methyltransferase 1 (DNMT1) down-regulation, DNA demethylation and gene expression of Patched1 (PTCH1), and results in the suppression of epithelial-mesenchymal transition (EMT). Moreover, activated HSCs contribute to DNA methylation of PTCH1.

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Salvianolic acid B-induced microRNA-152 inhibits liver fibrosis by attenuating DNMT1-mediated Patched1 methylation - PubMed (original) (raw)