Expression profiling and structural characterization of microRNAs in adipose tissues of hibernating ground squirrels

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 miRNAs in brown (BAT) and white adipose tissue (WAT) during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six miRNAs including let-7a, let-7b, miR-107, miR-150, miR-222 and miR-31 was significantly downregulated in WAT (P<0.05), which was 16%-54% of euthermic non-torpid control squirrels, whereas expression of three miRNAs including miR-143, miR-200a and miR-519d was found to be upregulated by 1.32-2.34-fold. Similarly, expression of more miRNAs was downregulated in BAT during torpor. We detected reduced expression of 6 miRNAs including miR-103a, miR-107, miR-125b, miR-21, miR-221 and miR-31 (48%-70% of control), while only expression of miR-138 was significantly upregulated (2.91±0.8-fold of the control, P<0.05). Interestingly, miRNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas miRNAs with altered expression in BAT during torpor were linked to mitochondrial β-oxidation. miRPath target prediction analysis showed that miRNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase (MAPK) signaling, while the miRNAs upregulated in WAT were linked to transforming growth factor β (TGFβ) signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-miRNAs for the miRNAs used in this study, suggesting no structure-influenced changes in pre-miRNA processing efficiency in the squirrel. As well, the expression of miRNA processing enzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of miRNA expression in adipose tissues may be linked to distinct biological roles in WAT and BAT during hibernation and may involve the regulation of signaling cascades.

Keywords: Dicer; Ground squirrel; Hypometabolism; Non-coding RNA; Stress adaptation.

Supplementary Figure 1

Secondary structure of pre-miRNAs. Secondary structure of the pre-miRNAs examined in this study was predicted using the mfold software with default settings.

Supplementary Figure 2

Predicted co-regulation of genes in the MAPK signaling pathway targeted by the downregulated miRNAs in WAT during torpor. Genes targeted by miRNAs let-7a, let-7b, miR-107, miR-150, miR-222 and miR-31 are highlighted with a bolded yellow box.

Supplementary Figure 3

Predicted co-regulation of genes in the MAPK signaling pathway targeted by downregulated miRNAs in BAT during torpor. Genes targeted by miRNAs miR-103a, miR-107, miR-125b, miR-21, miR-221 and miR-31 are highlighted with a bolded yellow box.

Supplementary Figure 4

Predicted co-regulation of genes in the TGFβ signaling pathway targeted by upregulated miRNAs in WAT during torpor. Genes targeted by miRNAs miR-143, miR-200a and miR-519d are highlighted with a bolded red box.

Figure 1

miRNA expression in white adipose tissue during torpor Expression of miRNAs in white adipose tissue samples from the euthermic control (black bar) and torpid ground squirrels (gray bar) was evaluated by RT-PCR. Relative expression of indicated miRNAs was normalized to the expression of 5S rRNA from the same sample. The relative miRNA expression in the torpid animals was further normalized to that in the euthermic control, which was arbitrarily set as 1.0. Data are mean ± SEM (n = 4–5 independent trials from different animals). Significant difference in miRNA expression in torpid compared to that of the euthermic control according to Student’s t-test was indicated with ^∗(P < 0.05) or ^∗∗(P < 0.005).

Figure 2

miRNA expression in brown adipose tissue during torpor Expression of miRNAs in the brown adipose tissue samples from euthermic control (black bar) and torpid ground squirrels (gray bar) was evaluated by RT-PCR. Relative expression of indicated miRNAs was normalized to the expression of 5S rRNA from the same sample. The relative miRNA expression in the torpid animals was further normalized to that in the euthermic control, which was arbitrarily set as 1.0. Data are mean ± SEM (n = 4–5 independent trials from different animals). Significant difference in miRNA expression in torpid compared to that of the euthermic control according to Student’s t-test was indicated with ^∗(P < 0.05) or ^∗∗(P < 0.005).

Figure 3

Dicer protein expression during torpor A. Protein expression of Dicer in the white and brown adipose tissue samples from euthermic control (black bar) and torpid ground squirrels (gray bar) was examined using immunoblotting. B. Relative expression of Dicer was normalized to the expression of GAPDH from the same sample. The relative miRNA expression in the torpid animals was further normalized to that in the euthermic control, which was arbitrarily set as 1.0. Data are presented as mean ± SEM (n = 4 independent trials from different animals). Statistical analysis was performed using Student’s t-test.