Research Article: MiR-145 mediates zebrafish hepatic outgrowth through progranulin A signaling

Date Published: May 22, 2017

Publisher: Public Library of Science

Author(s): Ya-Wen Li, Keng-Yu Chiang, Yen-Hsing Li, Sung-Yu Wu, Wangta Liu, Chia-Ray Lin, Jen-Leih Wu, Zhiyuan Gong.


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.

Partial Text

The liver is the largest metabolic organ that maintains numerous vital functions in the body. The liver is composed of approximately 80% hepatocytes and other cell types, including cholangiocytes, Kupffer cells, stellate cells and sinusoidal endothelial cells [1]. Liver development is divided into three stages beginning with specification in the foregut endoderm and followed by hepatic differentiation and outgrowth. At the beginning of the specification stage, hepatoblasts are derived from endodermal cells for liver bud formation. Hepatoblasts then begin to differentiate into functional hepatocytes and cholangiocytes. Finally, hepatic outgrowth occurs as hepatocytes rapidly proliferate to the appropriate size for the liver [2, 3]. Many important transcription factors and growth factors including hhex, prox1, the hnf family, Fgfs, Bmps and the Wnt pathway are involved in these processes. For example, Hhex is expressed in the hepatic bud at 22–50 hours post-fertilization (hpf) [4]. Prox1 is also expressed at 22 hpf and persists through hepatic differentiation [5]. Hepatic nuclear factors (hnf1, 3, and 4) are abundantly expressed in the liver [3]. Many liver specific genes are transcriptionally activated by different HNFs that have been extensively identified for their roles in mammalian hepatogenesis. In addition, some signaling pathways mediating hepatogenesis are conserved in mammals and zebrafish. For example, hnf1b (also called vhnf1) is required for hepatic specification in zebrafish[6]. In the Wnt pathway, mutations in wnt2bb, which is expressed at 18–52 hpf, leading to delayed hepatic specification [7]. β-catenin activation is involved in hepatic differentiation and outgrowth, and is important for maintaining liver size [8].

The regulatory mechanisms of liver development are complex, and involve in many growth factors and transcription factors across three stages-hepatic specification, differentiation and outgrowth. The miRNAs are known to be important genetic regulators in development. A liver specific miRNA, miR-122a, has been reported to control hepatocyte differentiation [30]. Besides miR-122a, little is known about the function of other miRNAs involved in liver development. Researchers have generated liver-specific DICER1 knockout mice to prove that the loss of miRNAs increases liver cell proliferation. However, the fact that which miRNAs participate in embryonic liver cell proliferation remains unclear. In a previous study, PGRN is a pleiotropic autocrine growth factor that was found to be essential for hepatic outgrowth through transcriptional regulation of MET during liver development [16]. Therefore, we are interested in exploring the fine-tuning regulator on PGRN signaling involved in embryonic liver growth. We identified several miRNAs from the miRNA predictions of zebrafish GrnA. Because the 3’UTR of GrnA is 487 nucleotides in size, there are only putative four miRNAs, including miR-9, miR-206, miR-731 and miR-217 predicted by TargetScan. In this study, we first showed the dysregulation of four miRNAs couldn’t influence liver development. The miR-9 is abundantly expressed in developing neurogenic cell that regulates neuron differentiation [31]. The miR-206 is a muscle specific miRNA participating in myogenesis [32]. The miR-217 is expressed in the pancreas [33]. The miR-731 is induced by hypoxic stress [34]. Hence, the four miRNAs may not regulate liver development. We demonstrate that miR-145, which targets the CDS region of GrnA, can affect embryonic liver growth. The expression pattern of miR-145, especially in the embryonic liver, has not been confirmed. We also demonstrate an inverse correlation between miR-145 and GrnA expression during hepatogenesis. miR-145 expression in the liver primordium decreases from 30 to 96 hpf and GrnA expression increases from 30 to 96 hpf. The expression pattern of GrnA is similar to that previously reported [35]. miR-145 is expressed in liver of zebrafish embryos at 30 hpf. miR-145 is ubiquitously expressed at 19-somite zebrafish embryos as demonstrated by Zeng et al. [28]. Furthermore, we show that miR-145 is almost not expressed in zebrafish embryos at 96 hpf. Wienholds’s and Zeng’s groups have proved that miR-145 is expressed in gut but not in liver of zebrafish embryos at 96 hpf [28, 33]. Moreover, GrnA is widely expressed in many other tissues. Its expression increases from 24 to 96 hpf [35].