miR-34a-5p Increases Hepatic Triglycerides and Total Cholesterol Levels by Regulating ACSL1 Protein Expression in Laying Hens

Abstract

Accumulating evidence has shown that miR-34a serves as a posttranscriptional regulatory molecule of lipid metabolism in mammals. However, little studies about miR-34a on lipid metabolism in poultry have been reported until now. To gain insight into the biological functions and action mechanisms of miR-34a on hepatic lipid metabolism in poultry, we firstly investigated the expression pattern of miR-34a-5p, a member of miR-34a family, in liver of chicken, and determined its function in hepatocyte lipid metabolism by miR-34a-5p overexpression and inhibition, respectively. We then validated the interaction between miR-34a-5p and its target using dual-luciferase reporter assay, and explored the action mechanism of miR-34a-5p on its target by qPCR and Western blotting. Additionally, we looked into the function of the target gene on hepatocyte lipid metabolism by gain- and loss-of-function experiments. Our results indicated that miR-34a-5p showed a significantly higher expression level in livers in peak-laying hens than that in pre-laying hens. miR-34a-5p could increase the intracellular levels of triglycerides and total cholesterol in hepatocyte. Furthermore, miR-34a-5p functioned by inhibiting the translation of its target gene, long-chain acyl-CoA synthetase 1 (ACSL1), which negatively regulates hepatocyte lipid content. In conclusion, miR-34a-5p could increase intracellular lipid content by reducing the protein level, without influencing mRNA stability of the ACSL1 gene in chickens.

Keywords: ACSL1; chicken; liver; miR-34a-5p; total cholesterol; triglycerides.

Figure 1

Increased lipid metabolism occurs in the liver of peak-laying hens compared with that in pre-laying hens. (A) ELISA analysis of the concentrations of TG, T-CHO and VLDL in the serums of pre-laying (20 weeks old) and peak-laying (30 weeks old) hens (n = 6 for each group). * p < 0.05, ** p < 0.01; (B) the difference in the lipid droplet accumulation in the liver of pre-laying (20 weeks old) and peak-laying (30 weeks old) hens as determined by oil red O staining. Scale bar: 100 μm.

Figure 2

miR-34a-5p positively regulates hepatocyte TG and T-CHO levels in laying hens. (A) The relative expression of miR-34a-5p in the livers of pre-laying (20 weeks old) and peak-laying (30 weeks old) hens (n = 6); (B) detection of miR-34a-5p overexpression 24 h after transfecting miR-34a-5p mimics; (C) effects of overexpressed miR-34a-5p on intracellular TG and T-CHO levels in LMH cells; (D) detection of miR-34a-5p inhibition 24 h after transfecting miR-34a-5p inhibitor; (E) effects of miR-34a-5p inhibition on intracellular TG and T-CHO levels in LMH cells. All data are presented as mean ± SD (n = 4 to 6). * p < 0.05, ** p < 0.01.

Figure 3

Validation of the ACSL1 gene as a direct target of miR-34a-5p. (A) Complementary sequences of miR-34a-5p and the 3′ UTR of the ACSL1 gene among species. Red indicates the seed region of miR-34a-5p; blue indicates the target 3′ UTR of ACSL1 gene; (B) secondary structure of the RNA duplex of miR-34a-5p and the 3′ UTR of the ACSL1 gene. Red indicates the target 3′ UTR of the ACSL1 gene; green indicates miR-34a-5p; (C) validation of the interaction between miR-34a-5p and the ACSL1 3′ UTR by a dual-luciferase reporter system. Data are presented as mean ± SD (n = 3); (D) qRT-PCR analysis of ACSL1 mRNA abundance in LMH cells treated with miR-34a-5p mimics and control; (E) qRT-PCR analysis of ACSL1 mRNA in LMH cells treated with miR-34a-5p inhibitor and control; (F) Western blotting analysis of ACSL1 protein abundance in LMH cells treated with miR-34a-5p mimics, inhibitor and controls. The digit means the gray value ratio of the target band and the internal reference band. Data are presented as mean ± SD (n = 6) * p < 0.05, ** p < 0.01.

Figure 4

ACSL1 represses hepatocyte TG and T-CHO levels. (A) qRT-PCR analysis of ACSL1 mRNA expression in the livers of pre-laying (20 weeks old) and peak-laying (30 weeks old) hens; (B) Western blotting analysis of ACSL1 protein expression in the livers of pre-laying (20 weeks old) and peak-laying (30 weeks old) hens. The digit means the gray value ratio of the target band and the internal reference band; (C) detection of the ACSL1 overexpression by qRT-PCR at 24 h after the transfection of pcDNA3.1-ACSL1-EGFP; (D) ELISA analysis of intracellular TG and T-CHO levels in LMH cells after ACSL1 gene overexpression for 24 h; (E) detection of ACSL1 gene knockdown by qRT-PCR at 24 h after transfection with siACSL1; (F) ELISA analysis of intracellular TG and T-CHO levels in LMH cells after ACSL1 gene knockdown for 24 h. Data are presented as mean ± SD (n = 4 to 6). * p < 0.05, ** p < 0.01.