MicroRNA-26a/b and their host genes synergistically regulate triacylglycerol synthesis by targeting the INSIG1 gene

Abstract

The microRNA-26 (miR-26) family is known to control adipogenesis in non-ruminants. The genomic loci of miR-26a and miR-26b have been localized in the introns of genes encoding for the proteins of the C-terminal domain RNA polymerase II polypeptide A small phosphatase (CTDSP) family. Insulin-induced gene 1 (INSIG1) encodes a protein with a key role in the regulation of lipogenesis in rodent liver. In the present study, we investigated the synergistic function of the miR-26 family and their host genes in goat mammary epithelial cells (GMEC). Downregulation of miR-26a/b and their host genes in GMEC decreased the expression of genes relate to fatty acid synthesis (PPARG, LXRA, SREBF1, FASN, ACACA, GPAM, LPIN1, DGAT1 and SCD1), triacylglycerol accumulation and unsaturated fatty acid synthesis. Luciferase reporter assays confirmed INSIG1 as a direct target of miR-26a/b. Furthermore, inhibition of the CTDSP family also downregulated the expression of INSIG1. Taken together, our findings highlight a functional association of miR-26a/b, their host genes and INSIG1, and provide new insights into the regulatory network controlling milk fat synthesis in GMEC. The data indicate that targeting this network via nutrition might be important for regulating milk fat synthesis in ruminants.

Keywords: CTDSP family; INSIG1; TAG accumulation; microRNA-26.

Figure 1.

MiR-26a/b are expressed concomitantly with CTDSP1/2/L during lactation. (A) Analysis of miR-26a/b expression in dairy goat mammary gland tissue during different stages of lactation stages. (B) Analysis of CTDSP1/2/L expression in dairy goat mammary gland tissue during different stages of lactation stages. (A) and (B) were performed in quintuplicate and repeated 3 times (n = 15). (C) The correlation between expression levels of miR-26a/b and their host genes in 24 mid-lactation mammary gland of goats. Values are presented as means + standard error of the means, *, P < 0.05; **, P < 0.01.

Figure 2.

Induction of miR-26a/b and host genes expression by transcription factors. Cells were treated with Ad-PPARG/Ad-SREBF1 and T0901317 (T09), and then the miR-26a/b and their host genes expression levels was quantified by quantitative real-time PCR. Each treatment was carried out in triplicate and repeated 3 times (n = 9). Values are presented as mean ± SEM; *P < 0.05, **P < 0.01 vs Ad, ^#P < 0.05, ^##P < 0.01 vs DMSO.

Figure 3.

Changes in lipid formation and TAG content in GMEC caused by miR-26a/b and CTDSP family inhibition. GMEC were co-transfected with 120 nM oligonucleotides (60 nM each), and the lipid droplet and TAG level were measured at 48 h posttransfection. (A) Lipid droplet formation after oil red O treatment, scale bar: 200 μm. (B) Cellular TAG content. All experiments were performed in triplicate and repeated 3 times (n = 9). Values are presented as means ± SEM; a, b: significant difference among treatments in anti-miRNAs group (P < 0.05).

Figure 4.

MiR-26 family targets INSIG1. (A) The correlation between expression level of miR-26a/b and INSIG1 in 24 mid-lactation mammary gland tissue samples. (B) Regulation of INSIG1 mRNA level by miR-26a/b mimic. (C) Regulation of INSIG1 protein level by miR-26a/b mimic and inhibitor. (D) MiR-26 family site in INSIG1 3’UTR and the construction of the luciferase (Luc) expression vector fused to the INSIG1 3’UTR. Bta: Bovine; WT, Luc reporter vector with the WT INSIG1 3’UTR (2224 bp to 2612 bp); MU, Luc reporter vector with the mutation at miR-26 family site in INSIG1 3’UTR. (E) Induction or suppression of Luc activity with the WT or MU of the INSIG13’UTR by miR-26 family mimic or inhibitor. GMEC were co-transfected with 120 nM oligonucleotides (60 nM each), WT and MU. The Luc activity was measured at 48 h posttransfection. All experiments were performed in triplicate and repeated 3 times (n = 9). Values are presented as mean + SEM, *, P < 0.05; **, P < 0.01.