MiR-145 mediates zebrafish hepatic outgrowth through progranulin A signaling

<div><p>MicroRNAs (miRs) are mRNA-regulatory molecules that fine-tune gene expression and modulate both processes of development and tumorigenesis. Our previous studies identified progranulin A (GrnA) as a growth factor which induces zebrafish hepatic outgrowth through MET signaling. We also found that miR-145 is one of potential fine-tuning regulators of GrnA involved in embryonic hepatic outgrowth. The low level of miR-145 seen in hepatocarinogenesis has been shown to promote pathological liver growth. However, little is known about the regulatory mechanism of miR-145 in embryonic liver development. In this study, we demonstrate a significant decrease in miR-145 expression during hepatogenesis. We modulate miR-145 expression in zebrafish embryos by injection with a miR-145 mimic or a miR-145 hairpin inhibitor. Altered embryonic liver outgrowth is observed in response to miR-145 expression modulation. We also confirm a critical role of miR-145 in hepatic outgrowth by using whole-mount in situ hybridization. Loss of miR-145 expression in embryos results in hepatic cell proliferation, and vice versa. Furthermore, we demonstrate that GrnA is a target of miR-145 and GrnA-induced MET signaling is also regulated by miR-145 as determined by luciferase reporter assay and gene expression analysis, respectively. In addition, co-injection of GrnA mRNA with miR-145 mimic or MO-GrnA with miR-145 inhibitor restores the liver defects caused by dysregulation of miR-145 expression. In conclusion, our findings suggest an important role of miR-145 in regulating GrnA-dependent hepatic outgrowth in zebrafish embryonic development.</p></div

miR-145 modulates hepatocyte proliferation in zebrafish embryos.

<p>The liver sections of control mimic-, miR-145 inhibitor- and miR-145 mimic-injected fish were examined using PCNA staining at 4 dpf. The liver sections of three embryos per experimental group were used and three independent replicates were performed. miR-145 inhibitor-injected fish had more PCNA-positive hepatocytes (B, E) as compared to those in control (A,D). In contrast, a low signal was detected in response to miR-145 mimic treatment (C,F). The arrowhead indicates PCNA-positive hepatocytes. The percentage of PCNA-positive hepatocytes was counted (G). The blue staining is hematoxylin stain; I, intestine; L, liver; Scale bars, 20 μm; *P < 0.05, t-test.</p

GrnA rescues the hepatic outgrowth defect caused by miR-145 manipulation.

<p>Liver morphology was determined by EGFP expression at 4 dpf in <i>Tg (fabp10</i>:<i>EGFP)</i> embryos (A) or in embryos injected with control mimic (B), miR-145 inhibitor (C), miR-145 mimic (D), miR-145 inhibitor with <i>grnA</i> MO (0.25 ng/embryo) (E) and miR-145 mimic with <i>grnA</i> mRNA (0.4 ng/embryo) (F). Thirty embryos per experimental group were used and three independent replicates were performed. A 3D image of the liver was observed using Leica SP5 confocal microscope and Imaris software. The liver size was examined by measuring the volume of EGFP expression (G). Ten embryos per experimental group were used and three independent replicates were performed. Scale bars, 100 μm; EGFP, enhanced green fluorescent protein; *P < 0.05, t-test.</p

miR-145 and GrnA expression patterns are inversely correlated during liver development.

<p>(A,B) The expression patterns of miR-145 (A), <i>grnA</i> (B) and the hepatoblast-specific marker gene <i>prox1</i> at 30, 50, 72 and 96 hpf were examined using FISH in wild-type zebrafish embryos. Scale bars, 25 μm (at 30 and 50 hpf), 50 μm (at 72 and 96 hpf). The dotted circles indicate <i>prox1</i> positive cells that represent liver. Ten embryos per experimental group were used and three independent replicates were performed. (C) The fetal liver at 72 and 96 hpf were isolated to quantify liver specific expression patterns of miR-145 and <i>GrnA</i>. Fifteen fetal livers per experiment were used and three independent replicates were performed. The relative expression is normalized with internal control, U6 and <i>ef1a</i> expression.</p

Additional file 3: Figure S1. of Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection

Endogenous gene expressions of fatty acid synthesis genes were determined by real-time qPCR. The endogenous fatty acid synthesis genes, Fadsd2 (Fatty acid desaturase delta 2), Elovl2 (Elongase 2) and Elovl5 (Elongase 5) of zebrafish liver and muscle were analysis. (JPEG 301 kb

miR-145 is one of miRNAs prediction on zebrafish GrnA that affects embryonic liver growth.

<p>(A) The miRNAs prediction of GrnA. (B) Liver morphology at 4 dpf was analyzed after control mimic, miR-145 mimic, miR-9 mimic, miR-206 mimic, miR-731 mimic and miR-217 mimic injections in <i>Tg(fabp10</i>:<i>EGFP)</i> embryos. The EGFP expression of <i>Tg (fabp10</i>:<i>EGFP)</i> fish showed a smaller liver phenotype in miR-145 mimic-treated fish compared to wild type, control fish and other miRNAs mimic-treated fish. Thirty embryos per experimental group from one clutch were used and three independent replicates were performed. (C) A 3D image of the liver was analyzed using Leica SP5 confocal microscope and Imaris software. The liver size was examined by measuring the volume of EGFP expression. Ten embryos per experimental group were used and three independent replicates were performed. Scale bars, 100 μm; EGFP, enhanced green fluorescent protein; *P < 0.05, t-test.</p

miR-145 is required for hepatic outgrowth.

<p>A WISH assay was used for determination of the liver developmental markers <i>hhex</i> at 30 hpf (A-C, dorsal view), <i>sePb</i> at 50 hpf (D-F, dorsal views), <i>prox1</i> at 72 hpf (G-I, dorsal views) and <i>fabp10</i> at 96 hpf (J-L, dorsal views) in control, miR-145 inhibitor-injected and miR-145 mimic-injected embryos. The arrowhead indicates liver. The expression levels of <i>hhex</i>, <i>sePb</i>, <i>prox1</i> and <i>fabp10</i> were examined using qPCR (M). The <i>ef1a</i> expression level was served as an internal control (*P < 0.05, t-test). Twenty whole embryos per experimental group were used and three independent replicates were performed.</p

miR-145 regulates GrnA and MET gene expression.

<p>The expression levels of the <i>grnA</i> and <i>met</i> genes were examined using qPCR after 24 hours of control, miR-145 mimic and miR-145 inhibitor treatment in ZFL cells (A). The protein levels of GrnA, MET and GAPDH were examined by Western blotting in ZFL cells at 48 hours after control, miR-145 mimic and miR-145 inhibitor treatments. The relative GrnA, MET and GAPDH protein levels were quantified as shown in the low panel (*P < 0.05, t-test) (B). Three independent replicates were performed.</p

miR-145 directly targets grnA as determined by luciferase assay.

<p>The predicted miR-145 target site on GrnA CDS is illustrated. WT and mutant forms of <i>GrnA</i> CDS were constructed in <i>psi-check2</i> reporter vector (A). miR-145 mimic and <i>psi</i>-grna-WT/<i>psi</i>-grna-mutant vector were co-transfected into ZFL cells for the luciferase assay. The luciferase activity of <i>psi</i>-grna-WT was suppressed approximately 45% in response to miR-145 mimic treatment. In contrast, the luciferase activity of <i>psi</i>-grna-mutant was unchanged in response to miR-145 mimic treatment (*P < 0.05, t-test) (B). Three independent replicates were performed.</p

Liver-Specific Expressions of <i>HBx</i> and <i>src</i> in the <i>p53</i> Mutant Trigger Hepatocarcinogenesis in Zebrafish

<div><p>Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of <i>src</i> in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and <i>src</i> transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential <i>in vivo</i> platforms for drug screening for therapies for human liver cancer.</p> </div