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MicroRNA125b-mediated Hedgehog Signaling Influences Liver Regeneration by Chorionic Plate-Derived Mesenchymal Stem Cells

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MicroRNA125b-mediated Hedgehog Signaling Influences Liver Regeneration by Chorionic Plate-Derived Mesenchymal Stem Cells

Jeongeun Hyun et al. Sci Rep.

Abstract

Although chorionic plate-derived mesenchymal stem cells (CP-MSCs) were shown to promote liver regeneration, the mechanisms underlying the effect remain unclear. Hedgehog (Hh) signaling orchestrates tissue reconstruction in damaged liver. MSCs release microRNAs mediating various cellular responses. Hence, we hypothesized that microRNAs from CP-MSCs regulated Hh signaling, which influenced liver regeneration. Livers were obtained from carbon tetrachloride (CCl4)-treated rats transplanted with human CP-MSCs (Tx) or saline (non-Tx). Sonic Hh, one of Hh ligands, increased in CCl4-treated liver, whereas it decreased in CP-MSC-treated liver with CCl4. The expression of Hh-target genes was significantly downregulated in the Tx. Reduced expansion of progenitors and regressed fibrosis were observed in the liver of the Tx rats. CP-MSCs suppressed the expression of Hh and profibrotic genes in co-cultured LX2 (human hepatic stellate cell) with CP-MSCs. MicroRNA-125b targeting smo was retained in exosomes of CP-MSCs. CP-MSCs with microRNA-125b inhibitor failed to attenuate the expression of Hh signaling and profibrotic genes in the activated HSCs. Therefore, these results demonstrated that microRNA-125b from CP-MSCs suppressed the activation of Hh signaling, which promoted the reduced fibrosis, suggesting that microRNA-mediated regulation of Hh signaling contributed to liver regeneration by CP-MSCs.

Figures

Figure 1
Figure 1. Hh ligands, Shh and Ihh, are differentially expressed in liver of Tx rats.
(A,B) Western blot analysis for Shh (27 kDa), Ihh (19 kDa: processed form/42 kDa: precursor form) and Gapdh (36 kDa, as an internal control). Data shown represent one of three experiments with similar results ((A) immunoblot/(B) band intensity). The mean ± SD results obtained by measuring the band density of three different blots are graphed (*p < 0.05, **p < 0.005 vs. control). (C) Immunohistochemistry for Shh and Ihh in liver sections from representative non-Tx and Tx rats at two weeks post transplantation (×40). Shh-positive and -negative hepatocytes are indicated by arrowhead and arrow, respectively. (D) Magnified image of Ihh-positive hepatic oval- and stellate-looking cells indicated by arrowhead and arrow, respectively (×100).
Figure 2
Figure 2. Downregulation of Hh activator, Smo, and Hh target gene, Gli3, in the CP-MSC-transplanted livers.
(A) QRT-PCR of smo and gli3 in the livers from the healthy (CON), non-Tx and Tx group. Medians and ranges of results are graphed (*p < 0.05 vs. CON). (B & C) Western blot analysis for Smo (86 kDa), Gli3FL (full-length, 190 kDa) and Gli3R (repressor form, 83 kDa). Gapdh (36 kDa) expression was used as an internal control (n = 4/group). Data shown represent one of three experiments with similar results ((B) immunoblot/(C) band intensity). The mean ± SD results obtained by measuring the band density of three different blots are graphed (*p < 0.05, **p < 0.005 vs. CON). (D) Immunohistochemical staining for Gli3 in liver sections from the representative non-Tx and Tx rats at two and three weeks post transplantation (×40). The inserted image shows the representative liver section from the CON.
Figure 3
Figure 3. Reduced fibrosis in Tx rats.
(A) QRT-PCR analysis for the profibrotic molecules including tgf-β, a fibrosis-stimulating factor, and α-sma, a fibrotic molecule, in the livers from the non-Tx, Tx and control (CON) group. Medians and ranges of results are graphed (*p < 0.05 vs. CON). (B & C) Western blot analysis for Tgf-β (25 kDa), α-SMA (42 kDa) and Gapdh (36 kDa, internal control) (n = 4/group). Data shown represent one of three experiments with similar results ((B) immunoblot/(C) band intensity). The mean ± SD results obtained by measuring the band density of three different blots are graphed (*p < 0.05, **p < 0.005 vs. CON). (D) Hepatic hydroxyproline content in all rats (n ≥ 4/group). The results are showed as the mean ± SEM (*p < 0.05, **p < 0.005 vs. CON). (E) Immunohistochemical staining for α-SMA (×40) and sirius red staining (×10) for examining collagen deposition were performed in liver sections from the representative non-Tx and Tx rat at two weeks.
Figure 4
Figure 4. CP-MSCs suppress activation of hedgehog signaling and expression of profibrotic genes, and reduce cell viability of LX2.
(A) QRT-PCR of the expression of Hh signals, including shh, smo, ptc, and gli3, and profibrotic genes, including tgf-β, col1α1, and vimentin, in LX2 mono-culture (alone, broken line), co-cultured LX2 with CP-MSCs (CP-MSC, solid line), and an LX2 mono-culture treated with 1 μM GDC-0449, an Smo antagonist (GDC-0449, dotted line), for 12 hours or 36 hours. The treatment time point was indicated as zero time. The level of gene expression in LX2 treated as above was shown as fold increase or decrease compared to that in fully activated LX2 at 48 hour (Supplementary Fig. S6). The mean ± SEM results obtained from three repetitive experiments are graphed (*p < 0.05, **p < 0.005 vs. mono-culture). (B) Cell number (growth) of mono-cultured LX2, co-cultured LX2 with CP-MSCs, and mono-cultured LX2 treated with 1 μM GDC-0449 for 12 or 36 hours was analyzed using the PrestoBlue™ assay. The mean ± SEM results obtained from three repetitive experiments are graphed (*p < 0.05, **p < 0.005 vs. mono-culture).
Figure 5
Figure 5. MiRNA-125b is expressed in CP-MSCs.
(A) QRT-PCR analysis of the expression of miRNA-125b, miRNA-324-5p and miRNA-326 in human normal liver (Normal), LX2 (human HSC line) and human CP-MSCs cultured during 72 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (*p < 0.05, **p < 0.005 vs. Normal). (B) In situ hybridization (ISH) with miRNA-125b probe (50 nM) in CP-MSCs and LX2 cultured for 24 hours. ISH with scrambled-miRNA probe (50 nM) in CP-MSCs as a negative control (NC). (C) QRT-PCR analysis of hepatic expressions of miRNA-125b in the livers from all rats. Medians and ranges of results are graphed (*p < 0.05 vs. CON). (D) Representative western blot analysis for CD9 (22 or 24 kDa), an exosomal marker, and GAPDH (36 kDa) in lysates of normal human liver (normal), LX2, and CP-MSCs (Cell lysate) and exosomes from CP-MSC-CM (Exosomes). (E) QRT-PCR analysis for miRNA-125b expression in Normal liver, LX2, CP-MSC lysates, and exosomes produced by CP-MSCs. The mean ± SEM results obtained by three repetitive experiments are graphed (*p < 0.05).
Figure 6
Figure 6. MiRNA-125b-downregulated CP-MSCs fail to downregulate expression of Hh signaling and profibrotic genes in LX2.
QRT-PCR analysis of the expression of Hh signals, including shh, smo, gli2, and gli3, and profibrotic genes, including vimentin and mmp9, in LX2 mono-culture (alone), LX2 treated with 1 μM GDC-0449 (Smo antagonist), and co-cultured LX2 with CP-MSCs (CP-MSC), CP-MSC having miRNA-125b inhibitor (CP-MSC miRNA-125b inhibitor), or CP-MSC having scrambled-miRNA inhibitor (CP-MSC (-) CON) for 12 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (*p < 0.05, **p < 0.005 vs. LX2 alone).
Figure 7
Figure 7. Hh signaling and profibrotic genes were highly expressed in activated primary HSCs co-cultured with CP-MSCs containing miRNA-125b inhibitor.
QRT-PCR analysis of the expression of Hh signals, including shh, smo, gli2, and gli3, and profibrotic genes, including vimentin and mmp9, in mono-cultured primary HSCs isolated from rats with CCl4-induced fibrosis (alone), and co-cultured primary rat HSCs with CP-MSCs (CP-MSC), CP-MSC having miRNA-125b inhibitor (CP-MSC miRNA-125b inhibitor), or CP-MSC having scrambled-miRNA inhibitor (CP-MSC (-) CON) for 12 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (*p < 0.05, **p < 0.005 vs. pHSC alone).
Figure 8
Figure 8. MiRNA-125b regulates expression of Hh signaling and profibrotic genes in LX2.
(A) QRT-PCR analysis of miRNA-125b expression in LX2 or transfected LX2 with miRNA-125b mimic (25 nM) or scrambled-miRNA mimic ((-) CON, 25 nM) as a negative control for 24 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (**p < 0.005). (B) QRT-PCR analysis for genes of Hh signaling, including shh, smo, gli2 and gli3, and profibrotic genes, including col1α1, vimentin and mmp9, in LX2 transfected with either scrambled-miRNA mimic ((-) CON) or miRNA-125b mimic for 24 hours. The mean ± SEM results obtained from three repetitive experiments are graphed (**p < 0.05 vs. (-) CON).

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