Induction of microRNA-214-5p in human and rodent liver fibrosis - PubMed (original) (raw)
Induction of microRNA-214-5p in human and rodent liver fibrosis
Masashi Iizuka et al. Fibrogenesis Tissue Repair. 2012.
Abstract
Background: miRNAs are non-coding RNAs that regulate gene expression in a wide range of biological contexts, including a variety of diseases. The present study clarified the role of miR-214-5p in hepatic fibrogenesis using human clinical tissue samples, livers from rodent models, and cultured hepatic stellate cells.
Methods: The expression of miR-214-5p and genes that are involved in liver fibrosis were analyzed in hepatitis C virus-infected human livers, rodent fibrotic livers, a human stellate cell line (LX-2), and the cells from intact mouse livers using real-time PCR. The effect of miR-214-5p overexpression in LX-2 cells on cell function was investigated. Twist-1 expression in the liver tissues of mouse models and primary-cultured stellate cells was also analyzed.
Results: miR-214-5p was upregulated in human and mouse livers in a fibrosis progression-dependent manner. miR-214-5p expression increased during the culture-dependent activation of mouse primary stellate cells and was significantly higher in stellate cells than in hepatocytes. The overexpression of miR-214-5p in LX-2 cells increased the expression of fibrosis-related genes, such as matrix metalloproteinase (MMP)-2, MMP-9, α-smooth muscle actin, and transforming growth factor (TGF)-β1. TGF-β stimulation induced miR-214-5p in LX-2 cells. Twist-1 was increased in fibrotic mouse livers and induced during mouse stellate cell activation.
Conclusion: miR-214-5p may play crucial roles in the activation of stellate cells and the progression of liver fibrosis. Twist-1 may regulate miR-214-5p expression in the liver, particularly in stellate cells.
Figures
Figure 1
miR-214-5p expression in the livers of patients with chronic hepatitis C virus infection. miR-214-5p expression in the livers of 35 hepatitis C virus (HCV)-infected patients was analyzed using real-time PCR. (A) Representative sirius red staining of liver tissues with fibrosis stages F1-F4 of chronic hepatitis C patients included in this study. (B) miR-214-5p expression in samples from each stage of liver fibrosis (METAVIR scoring system). The numbers of patients in each stage were: F1, 17; F2, 8; F3, 8; and F4, 2. The Jonckheere-Terpstra test identified trends among classes. (C) Comparison of miR-214-5p expression in F1/F2 and F3/F4 samples. (D) Comparison of miR-214-5p expression between F1 and F2-4. The levels of miR-214-5p expression in B, C and D are indicated relative to F1, F1/F2 and F1, respectively. *P < 0.05.
Figure 2
miR-214-5p expression in mouse livers with fibrosis induced by an methionine- and choline-deficient diet. (A) Sirius red staining (left and middle panels) and α-smooth muscle actin (SMA) immunostaining (right panels) of mouse liver tissues. Collagen deposition and an increase in α-SMA-positive cells were evident around the central vein area of the liver of mice that received the MCDD for 15 weeks. Scale bars, 100 μm (left and middle panels) and 200 μm (right panels). P, portal vein. C, central vein. (B) The mRNA expression of α-smooth muscle actin (α-SMA), the type 1 collagen alpha 1 chain (Col1a1), platelet-derived growth factor receptor (PDGFR)-β, transforming growth factor (TGF)-β1, fibronectin (FN)1, discoidin domain receptor (DDR)2, and β1 integrin (ITGB1) in fibrotic mouse livers was analyzed using real-time PCR. The results are expressed relative to mRNA expression at 5 weeks of the methionine- and choline-control diet (MCCD). *P < 0.05. **P < 0.01. (C) miR-214-5p expression in fibrotic mouse livers was analyzed using real-time PCR. The results are expressed relative to the expression of miR-214-5p at 5 weeks of the MCCD. *P < 0.05. **P < 0.01.
Figure 3
miR-214-5p expression in rat livers with fibrosis induced by a methionine- and choline-deficient diet. Expression of miR-214-5p in methionine- and choline-deficient diet (MCDD)-induced liver fibrosis and recovery phase in rats. The results are expressed relative to the expression of miR-214-5p in the livers of rats fed the methionine- and choline-control diet (MCCD) for 10 weeks. MCCD; rats fed MCCD for 10 weeks. MCDD; rats fed MCDD for 10 weeks. Recover; rats fed MCDD for 8 weeks followed by MCCD for 2 weeks. **P < 0.01.
Figure 4
miR-214-5p expression in liver cells, including stellate cells. (A) miR-214-5p expression in mouse stellate cells during primary culture was analyzed using real-time PCR. Stellate cells were isolated from mouse livers and cultured for 1, 4 or 7 days. The results are expressed relative to the expression of miR-214-5p at day 1. *P < 0.05. (B) The expression of α- smooth muscle actin (α-SMA), the type 1 collagen alpha 1 chain (Col1a1), platelet-derived growth factor receptor (PDGFR)-β, fibronectin (FN)1 and transforming growth factor (TGF)-β1 mRNA in primary-cultured mouse stellate cells was analyzed using real-time PCR. The results are expressed relative to the expression of the same mRNA at day 1. **P < 0.01. (C) miR-214-5p expression in HepG2, Huh7 and LX-2 cells was analyzed using real-time PCR. The results are expressed relative to the expression of miR-214-5p in HepG2. **P < 0.01. (D) miR-214-5p expression in hepatocytes (Hc), the non-parenchymal cell (NPC) fraction, and the hepatic stellate cell (HSC) fraction. The cells were isolated from intact mouse livers. The miR-214-5p expression in Hc, the NPC fraction, and the HSC fraction was analyzed using real-time PCR. The results are expressed relative to the expression of miR-214-5p in hepatocytes. **P < 0.01.
Figure 5
Effect of miR-214 overexpression on mRNA expression in LX-2 cells. (A) LX-2 cells were transfected with a miR-214 precursor or a negative control (control) at a final concentration of 50 nM and incubated for 24 hours. miR-214 expression was quantitated using real-time PCR. (B) The expression of fibrosis-related genes in LX-2 cells transfected with miR-214 precursors was analyzed using real-time PCR. The results are expressed as the expression relative to that in cells transfected with the control. **P < 0.01.
Figure 6
Regulation of miR-214-5p expression. (A) The effect of transforming growth factor (TGF)-β1 on miR-214-5p expression. LX-2 cells were treated with recombinant human TGF-β1 (3 or 10 ng/ml) for 24 hours in DMEM containing 0.1% fetal bovine serum (FBS). The results are expressed relative to miR-214 expression in cells that did not receive TGF-β1 treatment. *P < 0.05. (B) Twist-1 expression in the fibrotic livers of mice fed a methionine- and choline-deficient diet (MCDD). Twist-1 expression was analyzed using real-time PCR. The results are expressed relative to Twist-1 expression in methionine- and choline-control diet (MCCD) mice. *P < 0.05. (C) Twist-1 expression in primary-cultured mouse stellate cells. Twist-1 expression was analyzed using real-time PCR. The results are expressed relative to Twist-1 expression in cells on day 1. *P < 0.05, **P < 0.01.
References
LinkOut - more resources
Full Text Sources
Miscellaneous