Hydatid cyst fluid promotes peri-cystic fibrosis in cystic echinococcosis by suppressing miR-19 expression

Abstract

Background: Echinococcus granulosus infection causes cystic echinococcosis (CE); the generation of liver fibrosis around the parasitic larval cyst (metacestode) may play a major role in the spontaneous limitation of the parasitic growth; however, fibrogenesis has received little attention in CE. It has been reported that miR-19b plays a role in various diseases, including infectious diseases, by regulating fibrogenesis. However, its function in the development of liver fibrosis in E. granulosus infection is unknown.

Methods: The expression of miR-19b and genes that are involved in liver fibrosis were analysed in E. granulosus-infected human livers using qRT-PCR. The role of miR-19b on hepatic stellate cells (LX-2 cells in vitro) treated with hydatid cyst fluid (HCF) was then analysed by 3-(4, 5-dimet-hylthiazol-2-yl)-2, 4-diphenyl-tetrazolium bromide (MTT) assay, qRT-PCR, Western blot and flow cytometry.

Results: The results showed that the expression of miR-19 was significantly reduced in the pericystic collagen-rich liver tissue of CE patients, compared to normal liver. Incubation of LX-2 cells (in vitro) with HCF induced a decreased proliferation of these cells and a reduced expression of miR-19, inversely correlated with the expression of collagen 1A1 and TGF-β receptor II (TβRII). Conversely, overexpression of miR-19 by LX-2 cells inhibited the proliferation of these cells and led to decreased TβRII expression.

Conclusions: Our study provides new evidence for the intervention of miRNAs in the regulation of fibrosis in infectious diseases; it suggests that E. granulosus can inhibit miR-19 liver expression and promote fibrosis through the increase in TβRII, the activation of hepatic stellate cells and extracellular matrix production.

Keywords: Echinococcus granulosus; HSC; Liver fibrosis; TβRII; miR-19.

Fig. 1

Levels of α-SMA, COL1A1, COL3A1, TβRII mRNA and miR-19b expression in patients with Cystic Echinococcosis (CE) and correlation between α-SMA, COL1A1, COL3A1 or TβRII mRNA expression and miR-19b. a Levels of α-SMA mRNA in patients with CE. b Levels of COL1A1 mRNA in patients with CE. c Levels of COL3A1 mRNA in patients with CE. d Levels of TβRII mRNA in patients with CE. e Levels of miR-19b in patients with CE. f Correlation between miR-19b expression and α-SMA mRNA expression. g Correlation between miR-19b expression and COL1A1 mRNA expression. h Correlation between miR-19b expression and COL3A1 mRNA expression. i Correlation between miR-19b expression and TβRII mRNA expression. Normal: liver parenchyma distant from parasitic cyst; ‘Close’: liver parenchyma close to parasitic cyst. *P < 0.05 vs Normal

Fig. 2

Effect of HCF on LX-2 cells proliferation and cell cycle. a The effect of HCF on LX-2 cells proliferation. b The effect of HCF on cell cycle in 48 h. Cells were treated with 125 μg/ml HCF for 48 h, as determined by FACS analysis. Control: the non-treated cells. *P < 0.05 vs Control

Fig. 3

Effect of HCF on the expression of miR-19b, α-SMA, COL1A1, COL3A1 and TβRII in LX-2 cells. a Expression of miR-19b in HCF treated LX-2 cells. b Expression of α-SMA mRNA in HCF treated LX-2 cells. c Expression of COL1A1 mRNA in HCF treated LX-2 cells. d Expression of COL3A1 mRNA in HCF treated LX-2 cells. e Expression of TβRII mRNA in HCF treated LX-2 cells. f Expression of α-SMA, COL1A1, COL3A1 and TβRII protein in HCF treated LX-2 cells for 48 h. g–j Western blot results were quantified using densitometry analysis. *P < 0.05 vs Control

Fig. 4

Effect of miR-19b overexpression on the proliferation and cell cycle distribution of HCF-induced LX-2 cells. a The effect of miR-19b overexpression on the proliferation of HCF-induced LX-2 cells by MTT assay. b The effect of miR-19b on cell cycle of HCF-induced LX-2 cells was analysed by flow cytometry. *P < 0.05

Fig. 5

Effect of miR-19b on the expression of α-SMA, COL1A1, COL3A1 and TβRII in LX-2 cells. LX-2 cells were transiently transfected with miR-19b mimics (50 nM) or miRNA negative control (miR-NC) and a, α-SMA; b, COL1A1; c, COL3A1; d, TβRII gene expression were assessed by QRT-PCR at 48 h. e, 48 h post-transfection cells were harvested and immunoblot performed on whole cell lysates for α-SMA, COL1A1, COL3A1 and TβRII. Relative amount of f, α-SMA; g, COL1A1; h, COL3A1; i, TβRII expression were quantified using densitometry analysis. *P < 0.05