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MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression

Nature Communications (2016) 7
DOI: 10.1038/ncomms10993
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Abstract

Hedgehog (Hh) signalling regulates hepatic fibrogenesis. MicroRNAs (miRNAs) mediate various cellular processes; however, their role in liver fibrosis is unclear. Here we investigate regulation of miRNAs in chronically damaged fibrotic liver. MiRNA profiling shows that expression of miR-378 family members (miR-378a-3p, miR-378b and miR-378d) declines in carbon tetrachloride (CCl4)-treated compared with corn-oil-treated mice. Overexpression of miR-378a-3p, directly targeting Gli3 in activated hepatic stellate cells (HSCs), reduces expression of Gli3 and profibrotic genes but induces gfap, the inactivation marker of HSCs, in CCl4 -treated liver. Smo blocks transcriptional expression of miR-378a-3p by activating the p65 subunit of nuclear factor-κB (NF-κB). The hepatic level of miR-378a-3p is inversely correlated with the expression of Gli3 in tumour and non-tumour tissues in human hepatocellular carcinoma. Our results demonstrate that miR-378a-3p suppresses activation of HSCs by targeting Gli3 and its expression is regulated by Smo-dependent NF-κB signalling, suggesting miR-378a-3p has therapeutic potential for liver fibrosis.

Figures

  • Figure 1 | MiR-378 family was downregulated in injured livers of CCl4-treated mice. (a) Microarray analysis for miRNA expression was performed with total RNA extracted from livers of mice treated with corn-oil (control; CON) or CCl4 (n¼ 3 per group) for 10 weeks. Heat map shows the two-way
  • Figure 2 | Decreased expression of the miR-378 family in aHSCs. (a) Expression of miR-378 family members, including miR-378a-3p, miR-378b and miR-378d, in HepG2 (human liver epithelial cell line) and LX2 (human aHSC line) cells was examined by qRT–PCR. (b) qRT–PCR of the miR-378 family in primary qHSCs isolated from normal C57BL/6 mice at quiescent stage (d0; immediately after isolation) and in primary aHSCs (d7; cultured for 7 days). (c) qRT–PCR of the miR-378 family in primary
  • Figure 3 | MiR-378 binds directly to Gli3. (a) Using an miRNA database (www.miRNA.org), putative binding sites (red font) of miR-378a-3p were predicted in the 30-UTR of gli2 and gli3 mRNA in mouse and human. The dashed line represents complementary base pairs between miR-378a-3p and gli2 or gli3 mRNA, whereas the grey shading indicates the seed sequence of miR-378a-3p. (b) psiCHECK-2 vectors containing either the wild-type (wt) or mutant (mut) binding site of miR-378a-3p in gli2 and gli3 mRNAs were constructed to conduct luciferase reporter assays. The mutated nucleotides are shown in Supplementary Fig. 6. (c) Dual luciferase reporter assay was performed to verify binding between miR-378a-3p and gli2 or gli3 mRNA. N2a, a mouse neuroblastoma cell line, was co-transfected with a psiCHECK-2 vector containing either the wt or mut target sites plus either the miR-378a-3p
  • Figure 4 | MiR-378a-3p induces inactivation of primary HSCs by reducing Gli3 and Gli2 expression. (a) Primary aHSCs were transfected with either an miR-378a-3p mimic (white bar) or scrambled (Scr)-miR (control) (black bar) oligonucleotide for 24 and 48 h, and expression of gli3 and gli2 was assessed by qRT–PCR. (b) Western blot analysis and (c) cumulative densitometric analyses for nuclear Gli3 (145 kDa) and Gli2 (133 kDa), with Laminb1 (68 kDa) as an internal control for nuclear fraction. Data shown represent one of three experiments with similar results. (d) qRT–PCR analysis for genes related to activation of HSC, including vimentin, a-sma, col1a1 and mmp9, and the inactivation marker of HSC, gfap, in primary HSCs transfected with miR-378a-3p mimic (white bar) or scrambled (Scr)-miR (control) (black bar) oligonucleotide for 24 and 48 h. All results of relative expression values are shown as mean±s.e.m. obtained from triplicate experiments (unpaired two-sample Student’s t-test, *Po0.05 and **Po0.005 versus Scr-miR).
  • Figure 5 | Smo influences expression of miR-378. (a) qRT–PCR of the expression of Hh signals, including smo, gli2, gli3, gli1 and ptc, and profibrotic genes, including tgf-b, a-sma and col1a, in LX2 cells treated with (white bar) or without (black bar) 1 mM of GDC-0449, a Smoothened (Smo) antagonist, for 12, 24 and 48 h. (b) qRT–PCR analysis of miR-378a precursor (pri-miR-378a), miR-378b precursor (pri-miR-378b) and miR-378d precursor (pri-miR-378d) in LX2 cells treated with (white bar) or without (black bar) GDC-0449 for 12, 24 and 48 h. (c) qRT–PCR analysis of mature family members, including miR-378a3p, miR-378b and miR-378d, in LX2 cells treated with (white bar) or without (black bar) GDC-0449 for 12, 24 and 48 h. All results of relative expression values are shown as mean±s.e.m. obtained from triplicate experiments (unpaired two-sample Student’s t-test, *Po0.05 and **Po0.005 versus vehicle).
  • Figure 6 | Expression of miR-378a is regulated by Smo-dependent activation of p65. (a) p65 binding site (p65BS, grey box) on miR-378a DNA (arrow
  • Figure 7 | NPs having miR-378a-3p increase the level of miR-378a-3p but decrease Gli3 expression in CCl4-treated mice. (a) qRT–PCR for miR-378a-3p in livers from NP/NC, CCl4, CCl4-treated with NP/NC (CCl4þNP/NC) or NP/M (CCl4þNP/M) mice (n¼4 per group). Mean±s.e.m. results are graphed (Kruskal–Wallis test and unpaired two-sample Student’s t-test, *Po0.05 and **Po0.005 versus NP/NC). (b) qRT–PCR of miR-378a-3p in primary qHSCs isolated from normal mice at quiescent stage (d0; immediately after isolation), primary aHSCs (d7; cultured for 7 days) and primary HSCs isolated from CCl4þNP/M mice at 3 weeks after NPs treatment. Results of relative expression values are shown as mean±s.e.m. of triplicate experiments (one-way analysis of variance (ANOVA) with Tukey corrections, *Po0.05 and **Po0.005 versus d0). (c) qRT–PCR analysis for gli3 in livers from all mice at 3 weeks after NPs treatment (n¼4 per group). Mean±s.e.m. results are graphed (Kruskal–Wallis test and unpaired two-sample Student’s t-test, *Po0.05 and **Po0.005 versus NP/NC). (d) Western blot analysis for Gli3 (145 kDa) and Laminb1 (68 kDa, internal control for nuclear fraction) in livers of three
  • Figure 8 | MiR-378a-3p promotes inactivation of HSC in CCl4-treated mice. (a) qRT–PCR analysis for a-sma, vimentin, col1a1 and timp1 in NP/NC, CCl4, CCl4þNPs/NC and CCl4þNPs/M group (n¼4 per group). All results of relative expression values are shown as mean±s.e.m. (Kruskal–Wallis test and unpaired two-sample Student’s t-test, *Po0.05 and **Po0.005 versus NP/NC). (b) Western blot analysis for a-SMA (42 kDa), GFAP (50 kDa) and

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Hyun, J., Wang, S., Kim, J., Rao, K. M., Park, S. Y., Chung, I., … Jung, Y. (2016). MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression. Nature Communications, 7. https://doi.org/10.1038/ncomms10993

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