Short communicationLipolytic gene DAGLA is targeted by miR-223 in chicken hepatocytes
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.
Section snippets
Computational prediction of miR-223 target genes
The target genes of miR-223 and the binding sites between miR-223 and the 3′ UTR of the targets were predicted using the miRNA target prediction software miRDB, TargetScan and miRanda (Shao et al., 2014). The homology of target sites to other species were further analyzed by a BLAST search in GenBank (http://www.ncbi.nlm.nih.gov) (Wang et al., 2015b).
Plasmid construction
The 3′ UTR fragment of DAGLA containing the miR-223 binding site was amplified from chicken genomic DNA and inserted between XhoI and NotI sites
Prediction of miR-223 target genes
One hundred and thirty-eight, 168 and 134 genes were predicted as the target genes of miR-223 by TargetScan, miRDB and miRanda, respectively (Table S2). There were 349 target genes in total. Among them, DAGLA is a lipid metabolic gene. The 3′ UTR site of it was predicted to interact stably with miR-223, and the nucleotides 1–8 of miR-223 were completely complementary to the corresponding sequence (Fig. 1A). The 3′ UTRs of DAGLA in other species were also analyzed using BLAST search. Although
Discussion
Through bioinformatics, 349 genes were predicted as targets of miR-223. We verified DAGLA because it contains a nearly perfect target site for miR-223 and it is involved in lipid metabolism (Powell et al., 2015). DAGLA is a key enzyme in β-oxidation. It catabolizes diacylglycerol to monoacylglycerol. Thus, we think DAGLA may be involved in lipid metabolism in chicken liver. When we compared miR-223 in chickens with that in mice, humans and many other species, the sequence of miR-223 in chickens
Conclusions
The results of the present study support DAGLA as a target gene of miR-223 in chicken hepatocytes.
CRediT authorship contribution statement
Xingguo Wang: Conceptualization, Methodology, Investigation, Writing - original draft. Yongfeng Li: Investigation, Writing - review & editing. Liang Qu: Data curation, Visualization. Jun Guo: Formal analysis. Taocun Dou: Resources. Yuping Hu: Validation. Meng Ma: Validation. Kehua Wang: Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31601938), the Public Welfare Research Institute Independent Research of Jiangsu Province (BM2018026), the China Agriculture Research System (CARS-40-K01), and the Agricultural Major New Breed Creation of Jiangsu Province (PZCZ201729).
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These authors contributed equally to this work.