Expression of miR-33 from an SREBF2 intron targets the FTO gene in the chicken

Abstract

The sterol regulatory element binding transcription factor 2 (SREBF2) gene encodes a transcription factor that activates the expression of many genes involved in the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids. Through bioinformatics, we found that intron 16 of the chicken SREBF2 gene might encode the chicken miR-33. Using quantitative RT-PCR, we detected the expression of miR-33 in a variety of chicken tissues including skeletal muscle, adipose tissue, and liver. Three hundred and seventy eight genes were predicted to be potential targets of miR-33 in chickens via miRNA target prediction programs "miRanda" and "TargetScan". Among these targets, the gene FTO (fat mass and obesity associated) encodes a Fe(II)- and 2-oxoglutarate-dependent nucleic acid demethylase that regulates lipid metabolism, and the possibility that its expression is negatively regulated by miR-33 in the chicken liver was therefore further studied. Co-transfection and dual-luciferase reporter assays showed that the expression of luciferase reporter gene linked to the 3'-untranslated region (3'UTR) of the chicken FTO mRNA was down-regulated by overexpression of the chicken miR-33 in the C2C12 cells (P<0.05). Furthermore, this down-regulation was completely abolished when the predicted miR-33 target site in the FTO 3'UTR was mutated. In contrast, the expression of FTO mRNA in the primary chicken hepatocytes was up-regulated after transfection with the miR-33 inhibitor LNA-anti-miR-33. Using quantitative RT-PCR, we also found that the expression of miR-33 was increased in the chicken liver from day 0 to day 49 of age, whereas that of the FTO mRNA was decreased during the same age period. These data together suggest that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of the FTO gene.

Figure 1. Prediction of transcription of chicken miR-33 from the chicken SREBF2 gene.

A miR-33 stem-loop is predicted from intron 16 of SREBF2, and the sequence of this part of the SREBF2 gene is highly conserved across mammalian species (mmu: mouse; rno: rat; bta: cow; hsa: human) and chicken (gga: chicken).

Figure 2. Expression profile of miR-33 and SREBF2 mRNA in chicken tissues.

The expression levels of miR-33 and SREBF2 mRNA in 10 tissues from 4-wk-old chickens were analyzed by real-time qRT-PCR. The expression of miR-33 was normalized to 18S rRNA, and the expression of SREBF2 mRNA was normalized to β-actin mRNA. Data are means ± SEM (n = 3 chickens).

Figure 3. Construction of the pMIR-FTO and pMIR-FTOmut plasmids.

The top panel shows the structure and cloning sites of the pMIR-reporter vector. Wild type and the miR-33 binding site-mutated FTO 3′UTR were cloned into the reporter vector. The middle panel shows complementarity between miR-33 and predicted target site in the FTO 3′UTR. The bottom panel shows the sequences of either wild type or mutant FTO 3′UTR.

Figure 4. Verification of the interaction between miR-33 and the FTO 3′UTR.

A: Verification of over-expression of miR-33 in C2C12 cells. C2C12 cells were transfected with the control vector pcDNA3.1 or the miR-33 over-expression vector pcDNA3.1-miR-33. The expression level of miR-33 was detected by real-time qRT-PCR. Data are expressed as means ± SEM (n = 3). ** P<0.01. B: Reporter gene analysis of the interaction between miR-33 and FTO 3′UTR. C2C12 cells were co-transfected with pMIR-FTO or pMIR-FTOmut and pcDNA3.1 or pcDNA3.1-miR-33. Data are expressed as means ± SEM (n = 3). * P<0.05.

Figure 5. Effect of miR-33 knockdown on the expression of miR-33 and FTO mRNA in primary chicken hepatocytes.

A: Expression levels of miR-33. Primary chicken hepatocytes were transfected with LNA-anti-miR-33 or LNA scramble control. miR-33 and FTO mRNA were quantified by real-time qRT-PCR 48 h after transfection. Data are means ± SEM (n = 3), P = 0.4. B: Expression levels of FTO mRNA. Data are means ± SEM (n = 3), P = 0.2.

Figure 6. Expression levels of chicken miR-33 and FTO mRNA in chicken liver at different postnatal ages.

A. The expression levels of miR-33 and FTO mRNA in chicken liver from 0 to 49 d of ages were analyzed by qRT-PCR. The former was normalized to 18S rRNA, while the latter to β-actin mRNA. Data are means ± SEM (n = 3). B. Expression levels of chicken miR-33 and FTO mRNA in liver from 0 to 49 d of ages are negatively correlated (P = 0.07), as determined by a regression analysis.