miR-26a mediates LC-PUFA biosynthesis by targeting the Lxrα–Srebp1 pathway in the marine teleost Siganus canaliculatus

Abstract

MicroRNAs (miRNAs) have been recently shown to be important regulators of lipid metabolism. However, the mechanisms of miRNA-mediated regulation of long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis in vertebrates remain largely unknown. Herein, we for the first time addressed the role of miR-26a in LC-PUFA biosynthesis in the marine rabbitfish Siganus canaliculatus. The results showed that miR-26a was significantly down-regulated in liver of rabbitfish reared in seawater and in S. canaliculatus hepatocyte line (SCHL) incubated with the LC-PUFA precursor α-linolenic acid (ALA), suggesting that miR-26a may be involved in LC-PUFA biosynthesis due to its abundance being regulated by factors affecting LC-PUFA biosynthesis. Opposite patterns were observed in the expression of liver X receptor α (lxrα) and sterol regulatory element-binding protein-1 (srebp1), as well as the LC-PUFA biosynthesis related genes (Δ4 fads2, Δ6Δ5 fads2 and elovl5) in SCHL cells incubated with ALA. Luciferase reporter assays revealed rabbitfish lxrα as a target of miR-26a, and overexpression of miR-26a in SCHL cells markedly reduced protein levels of Lxrα, Srebp1 and Δ6Δ5 Fads2 induced by the agonist T0901317. Moreover, increasing endogenous Lxrα by knockdown of miR-26a facilitated Srebp1 activation and concomitant increased expression of genes involved in LC-PUFA biosynthesis, and consequently promoted LC-PUFA biosynthesis both in vitro and in vivo. These results indicate a critical role of miR-26a in regulating LC-PUFA biosynthesis through targeting the Lxrα-Srebp1 pathway and provide new insights into the regulatory network controlling LC-PUFA biosynthesis and accumulation in vertebrates.

Identification of miR-145 as a Key Regulator Involved in LC-PUFA Biosynthesis by Targeting hnf4α in the Marine Teleost Siganus canaliculatus

“…29,32 Furthermore, miR-33 and miR-24 can promote the biosynthesis of LC-PUFA by targeting insulin-induced gene 1 (insig1), thus facilitating the Srebp1 pathway, 31,33 and miR-26a mediates LC-PUFA biosynthesis through the liver X receptor α (Lxrα)-Srebp1 pathway by targeting lxrα. 34 These findings highlight the key roles of miRNAs in the post-transcriptional regulation of the metabolism of essential fatty acids in vertebrates. In the present study, we found that miR-145, like other identified miRNAs, including miR-33 and miR-24, 31,33 also responds to both salinity and availability of C 18 PUFA precursors (e.g.…”

“…Furthermore, as tissue distribution of miRNAs may partly reflect miRNA functions, 52 we examined the tissue distribution of miR-145 and found that miR-145 was ubiquitously expressed including in liver, while the expression level of hnf4α was relatively highest in intestine, followed by liver. 28 Further in silico analyses found that, among the LC-PUFA biosynthesis related genes, miR-145 potentially targeted the 3′UTR of hnf4α, which is different from other identified miRNAs' target genes, [29][30][31][32][33][34] and in vitro luciferase reporter assays identified hnf4α as a novel target of miR-145 in rabbitfish. Moreover, when knocked down the miR-145, the expression of Hnf4α and LC-PUFA biosynthesis key enzymes were significantly upregulated and subsequently the accumulation of LC-PUFA were increased both in hepatocytes in vitro and in rabbitfish in vivo.…”

“…Fish were injected into their abdominal cavity with 100 μL volume of total antagomirs diluted in PBS to 50 nmoL/mL twice weekly for 3 weeks. Fish were fed a commercial diet during the in vivo injection experiment, and the details of the fatty acid composition of the diet were described previously by Chen et al 34 At 21 d post-injection, the fish were fasted for 24 h and anesthetized with 0.01% 2-phenoxyethanol (Sigma-Aldrich), and then the eyes, brain, liver, and muscle tissues from each fish were collected, dipped immediately into liquid nitrogen, and stored at −80 °C for subsequent extraction of total RNA, proteins, and lipids.…”

“…[23][24][25] Moreover, we recently found that Hnf4α was also involved in regulating the biosynthesis of LC-PUFA in S. canaliculatus through transcriptionally regulating desaturase and elongase enzyme genes, including ∆4 fads2, Δ6Δ5 fads2and elovl5. [26][27][28] Furthermore, our recent work has shown that the expression of fads and elovl is also regulated directly or indirectly by microRNAs (miRNA or miR) at the posttranscriptional level in rabbitfish S. canaliculatus, [29][30][31][32][33][34] highlighting the vital regulatory roles of miRNAs in regulating biosynthesis of LC-PUFA in vertebrates. MiRNAs, small non-coding RNA molecules, regulate expression of genes through binding 3′ untranslated regions (3′UTR) of mRNAs post-transcriptionally, and they are now appreciated as key regulators of cell proliferation, differentiation, metabolism and inflammation.…”

“…35,36 Recently, miRNAs were shown to have impacts on lipid and lipoprotein metabolism, [37][38][39] and we have reported that certain miRNAs, namely miR-17, miR-24, miR-26a, miR-33 and miR-146a play pivotal roles in the regulation of LC-PUFA biosynthesis in the rabbitfish. [29][30][31][32][33][34] A further microRNA, miR-145, has been shown to be involved in cholesterol metabolism and adipogenesis in mammals. [40][41][42] For example, miR-145 increased cholesterol level in islets through decreasing ATP-binding cassette transporter A1 (ABCA1) expression in murine islets, 40 and attenuated lipolysis by directly targeting Foxo1 (forkhead box O1) and Cgi58 (comparative gene identification 58, also known as alpha/beta hydrolase domain 5, ABHD5) in white adipose tissue of mice.…”