Guanghui Chen1, Kun Wu1, Tao Zhao1, Shicheng Ling1, Wei Liu2, Zhi Luo1,3. 1. Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Fishery College, Huazhong Agricultural University, Wuhan, China. 2. Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China. 3. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
Abstract
BACKGROUND: microRNAs (miRNAs) post-transcriptionally regulate gene expression and act as important modulators of cholesterol homeostasis. OBJECTIVE: The study explores the mechanism by which miRNAs mediate high fat-induced changes of cholesterol metabolism in yellow catfish. METHODS: Yellow catfish (weight: 3.79 ± 0.16 g, 3 mo old, mixed sex) were fed 2 diets containing lipids at 11.3% [control (CON)] or 15.4% [high-fat diet (HFD)] (by weight) for 8 wk. Cholesterol content was measured; hematoxylin-eosin (H&E) staining, qPCR assays, and small RNA sequencing were conducted in the liver. Hepatocytes were isolated from separate, untreated fish and incubated for 24 h in control solution or palmitic acid (PA; 0.25 mM)/oleic acid (OA; 0.5 mM) after 4 h pretreatment with or without miR-144 inhibitor/mimic (40 nM). Cholesterol content was measured; qPCR assays and Western blotting were conducted in the hepatocytes. HEK293T cells were co-transfected with plasmids to validate miR-144 target genes. The promoter activities of miR-144 were analyzed in HEK293T cells with PA (0.25 mM) or OA (0.25 or 0.5 mM) treatment for 24 h. Luciferase activity assays, electrophoretic mobility shift assay, and Western blotting were conducted in HEK293T cells. RESULTS: Compared with CON, HFD induced hepatic cholesterol accumulation (31.5%), and upregulated miR-144 expression (8.40-fold, P < 0.05). miR-144 directly targeted hydroxymethylglutaryl-CoA reductase (hmgcr), cholesterol 7α-monooxygenase A1 (cyp7a1), and adenosine triphosphate binding cassette transporter A1 (abca1) in HEK293T cells. In the hepatocytes of yellow catfish, miR-144 inversely regulated the expression of hmgcr, cyp7a1, and abca1 (30.3-78.5%, P < 0.05); loss of miR-144 function alleviated PA- or OA-induced cholesterol accumulation (19.5-61.1%, P < 0.05). We also characterized the C/EBPα binding site in the miR-144 promoter, and found that C/EBPα positively regulated miR-144 expression through binding to the miR-144 promoter. CONCLUSIONS: miR-144 mediated HFD-induced changes in the liver and hepatocytes of yellow catfish, suggesting a possible mechanism for HFD-induced dysfunction in cholesterol metabolism.
BACKGROUND: microRNAs (miRNAs) post-transcriptionally regulate gene expression and act as important modulators of cholesterol homeostasis. OBJECTIVE: The study explores the mechanism by which miRNAs mediate high fat-induced changes of cholesterol metabolism in yellow catfish. METHODS:Yellow catfish (weight: 3.79 ± 0.16 g, 3 mo old, mixed sex) were fed 2 diets containing lipids at 11.3% [control (CON)] or 15.4% [high-fat diet (HFD)] (by weight) for 8 wk. Cholesterolcontent was measured; hematoxylin-eosin (H&E) staining, qPCR assays, and small RNA sequencing were conducted in the liver. Hepatocytes were isolated from separate, untreated fish and incubated for 24 h in control solution or palmitic acid (PA; 0.25 mM)/oleic acid (OA; 0.5 mM) after 4 h pretreatment with or without miR-144 inhibitor/mimic (40 nM). Cholesterolcontent was measured; qPCR assays and Western blotting were conducted in the hepatocytes. HEK293T cells were co-transfected with plasmids to validate miR-144 target genes. The promoter activities of miR-144 were analyzed in HEK293T cells with PA (0.25 mM) or OA (0.25 or 0.5 mM) treatment for 24 h. Luciferase activity assays, electrophoretic mobility shift assay, and Western blotting were conducted in HEK293T cells. RESULTS: Compared with CON, HFD induced hepatic cholesterol accumulation (31.5%), and upregulated miR-144 expression (8.40-fold, P < 0.05). miR-144 directly targeted hydroxymethylglutaryl-CoA reductase (hmgcr), cholesterol 7α-monooxygenase A1 (cyp7a1), and adenosine triphosphate binding cassette transporter A1 (abca1) in HEK293T cells. In the hepatocytes of yellow catfish, miR-144 inversely regulated the expression of hmgcr, cyp7a1, and abca1 (30.3-78.5%, P < 0.05); loss of miR-144 function alleviated PA- or OA-induced cholesterol accumulation (19.5-61.1%, P < 0.05). We also characterized the C/EBPα binding site in the miR-144 promoter, and found that C/EBPα positively regulated miR-144 expression through binding to the miR-144 promoter. CONCLUSIONS:miR-144 mediated HFD-induced changes in the liver and hepatocytes of yellow catfish, suggesting a possible mechanism for HFD-induced dysfunction in cholesterol metabolism.
Authors: Yan Wang; Peng Zhu; Jiahui Ni; Qilang Mo; Wei Luo; Zongjun Du; Jun Jiang; Song Yang; Liulan Zhao; Quan Gong Journal: Fish Physiol Biochem Date: 2022-03-01 Impact factor: 2.794