A Translational Model for Venous Thromboembolism: MicroRNA Expression in Hibernating Black Bears

Abstract

Background: Hibernating American black bears have significantly different clotting parameters than their summer active counterparts, affording them protection against venous thromboembolism during prolonged periods of immobility. We sought to evaluate if significant differences exist between the expression of microRNAs in the plasma of hibernating black bears compared with their summer active counterparts, potentially contributing to differences in hemostasis during hibernation.

Materials and methods: MicroRNA sequencing was assessed in plasma from 21 American black bears in summer active (n = 11) and hibernating states (n = 10), and microRNA signatures during hibernating and active state were established using both bear and human genome. MicroRNA targets were predicted using messenger RNA (mRNA) transcripts from black bear kidney cells. In vitro studies were performed to confirm the relationship between identified microRNAs and mRNA expression, using artificial microRNA and human liver cells.

Results: Using the bear genome, we identified 15 microRNAs differentially expressed in the plasma of hibernating black bears. Of these microRNAs, three were significantly downregulated (miR-141-3p, miR-200a-3p, and miR-200c-3p), were predicted to target SERPINC1, the gene for antithrombin, and demonstrated regulatory control of the gene mRNA expression in cell studies.

Conclusions: Our findings suggest that the hibernating black bears' ability to maintain hemostasis and achieve protection from venous thromboembolism during prolonged periods of immobility may be due to changes in microRNA signatures and possible upregulation of antithrombin expression.

Keywords: Anticoagulation; Antithrombin; Thrombosis; Venous thromboembolism; microRNA.

Fig. 1 –

Differentially expressed miRNAs (total 89) in the plasma of hibernating and active black. Red denotes hibernating black bears (n = 10; 40 miRNAs upregulated), and blue denotes active black bears (n = 11; 49 miRNAs upregulated) when comparing sequences to human genome using DESeq2 package in DEBrowser. (Color version of figure is available online.)

Fig. 2 –

Differential expressions of plasma miR-141–3p, miR200a-3p, and miR-200c-3p in hibernating and active black bears. Red denotes hibernating black bears (n = 10; downregulated miR-141–3p, miR200a-3p, and miR-200c-3p), and blue denotes active black bears (n = 11; upregulated miR-141–3p, miR200a-3p, and miR-200c-3p) using RSEM and DESeq2. (Color version of figure is available online.)

Fig. 3 –

SERPINC1 silencing in HuH7.5 cells after 48 h transfection measured using ddPCR, expressed as a ratio to human B2M and normalized to a control plasmid expressing an unrelated amiR. (Color version of figure is available online.)

Fig. 4 –

miRNAs regulating the hemostatic system. Demonstration of increased levels of circulating AT resulting from downregulated levels of miR-141–3p, miR-200a-3p, and miR-200c-3p and subsequent increased expression of SERPINC1 in hibernating American black bears. (Color version of figure is available online.)