Lipolytic gene DAGLA is targeted by miR-223 in chicken hepatocytes

Highlights

• DAGLA is targeted by miR-223 in chickens.

• The target site of miR-223 in chicken DAGLA is not conserved among species.

• DAGLA mRNA and protein are regulated by miR-223 in chicken hepatocytes.

Abstract

miR-223 is an important miRNA. It plays important roles in lipid metabolism by targeting related genes in mammals. It may be related to fatty liver in laying hens and its functions and target genes need further study. Through bioinformatics, we found that 349 genes were predicted as target genes of miR-223. Lipid-related gene DAGLA was among the predicted target genes. Dual-luciferase reporter assays showed that DAGLA was the target gene of miR-223 and the site mutation assays validated the target site of miR-223 in DAGLA. Overexpression of miR-223 in chicken hepatocytes LMH decreased the mRNA and protein expression of DAGLA, while knockdown of miR-223 increased expression of DAGLA in LMH cells, further indicating that miR-223 targets DAGLA and downregulates its expression. Since the target site of miR-223 in chicken DAGLA is not conserved, these findings suggest that miR-223 plays a specific role in chicken liver by regulating expression of target gene DAGLA.

Introduction

miRNAs are a class of small noncoding RNAs of approximately 22 nucleotides in length (Bartel, 2004). They negatively regulate gene expression by interacting with the 3′ untranslated region (UTR) of target mRNAs, causing translational repression or mRNA cleavage in animals (Lee et al., 1993, Yekta et al., 2004). miRNAs play roles in almost all biological processes, such as growth, development and metabolism (Bartel, 2004, Wienholds and Plasterk, 2005).

miR-223 was reported by Lim et al., (2003), who used bioinformatics to predict miR-223 genes in humans, mice and zebra fish. The presence of miR-223 in humans and mice was verified experimentally (Landgraf et al., 2007), and it has now been found to exist in various species. miR-223 is expressed in various tissues and its modes of expression are specific among different tissues. Its expression level increases in the differentiation process of myeloid cells (Gilicze et al., 2014), in the serum of colorectal cancer patients compared with healthy persons (Zheng et al., 2014), and in mouse liver with hepatic ischemia/reperfusion injury (Yu et al., 2009). Its expression level decreases in the differentiation process of monocytes and macrophages (Gilicze et al., 2014), in the serum (Giray et al., 2014) and hepatocytes (Wong et al., 2008) of hepatocellular carcinoma patients compared with healthy persons, and in osteogenic differentiation of preadipocytes (Du et al., 2019).

Originally, miR-223 was thought to take part in hematopoiesis only (Chen et al., 2004). Later, miR-223 was confirmed to be involved in lipid metabolism, carcinogenesis, inflammation, osteoblast differentiation and some other biological processes. In macrophages, overexpression of miR-223 inhibits Toll-like receptor signaling, slowing down lipid accumulation, while knockdown of miR-223 induces lipid accumulation (Wang et al., 2015a). In bladder cancer cells, overexpression of miR-223 slows down tumor progression and induces apoptosis by inhibiting target gene WDR62 (Sugita et al., 2019). In hepatoma cells, miR-223 induces resistance to sorafenib by inhibiting target gene FBW7 (Tang et al., 2019). In brain microglial cells, miR-223 inhibits autophagy by targeting ATG16L1, and then promotes inflammation of the central nervous system (Li et al., 2019). In bone marrow mesenchymal stem cells, knockdown of miR-223 promotes osteogenic differentiation by regulating target gene DHRS3 (Zhang et al., 2018).

In our previous study on chicken liver, we investigated the effect of growth hormone on liver metabolism, and found that it regulated some miRNAs and mRNAs; most of which were involved in lipid metabolism. miR-223 is among the upregulated miRNAs (Wang et al., 2014). We speculated that miR-223 played roles in lipid metabolism in chicken liver and targeted lipid metabolic genes. In the current study, we provide experimental evidence confirming that miR-223 targets lipid metabolic gene DAGLA and regulates its expression in chicken hepatocytes.