MiRNA regulation of auto-anticoagulation in hibernating black bears: A Novel Translational Approach

Hibernating American black bears have significantly different clotting parameters than their active summer counterparts, affording them innate protection against venous thromboembolism (VTE) despite prolonged periods of immobility. Physiologic changes that occur during hibernation are thought to result from differential gene expression, rather than novel genes, and there is increasing evidence miRNAs may play an important role this regulation. We propose that significant differences exist in miRNA expression in the plasma of hibernating black bears compared to their active counter parts (summer), which lead to critical gene regulation responsible for auto-anticoagulation during hibernation. Methods: Blood was collected from 21 American black bears in the Northern Michigan Peninsula in summer 2017 and winter 2018 (11 active, 10 hibernating). Plasma was extracted Results: Fifteen miRNAs were differentially expressed in the plasma of hibernating black bears. Nine miRNAs were significantly downregulated (miR10b-3p, miR-136-3p, miR-181c-5p , miR-200a-3p, miR-200b-5p, miR-200c-3p, miR-320b, miR-320c and miR-320d) and six miRNAs were significantly upregulated (miR-15a-5p, miR-15b-3p, miR-15b-5p, miR-16-5p, miR-92a-3p, miR-150-5p) during hibernation. Twelve miRNAs had no identifiable targets, but miR-200a-3p, miR-200b-5p and miR-200c-3p found to be targets of SERPINC1, the gene responsible for the production of antithrombin (AT). Conclusions: Several miRNAs were differentially expressed in hibernating bears (12). Most importantly miR-200a-3p, miR-200b-5p and miR-200c-3p were all downregulated in hibernation and associated with increased expression of SERPINC1 and production of AT. AT is a powerful anticoagulant and this finding may explain the hibernating black bears ability to achieve auto-anticoagulation and protection from VTE. We evaluated if significant differences exist between the expression of miRNAs in the plasma of hibernating black bears compared to their summer active counter parts. Please note that as some bears don't match due to unavailability of the same bears in summer time, differential expression analysis was carried out without pairing them.冬眠的美洲黑熊与夏季活跃状态下的同类相比，凝血参数存在显著差异，即便处于长时间静止状态，也能获得针对静脉血栓栓塞症（VTE）的先天防护能力。目前认为，冬眠过程中发生的生理变化源于差异基因表达，而非新基因的产生；且越来越多的证据表明，微RNA（microRNA, miRNA）在这一调控过程中发挥着重要作用。我们推测，与夏季活跃状态的同类相比，冬眠黑熊血浆中的miRNA表达存在显著差异，这些差异可通过调控关键基因实现冬眠期间的自身抗凝。 方法：研究于2017年夏季与2018年冬季，在密歇根半岛北部采集了21只美洲黑熊的血液样本（11只为活跃状态，10只为冬眠状态），并分离得到血浆。 结果：冬眠黑熊的血浆中共检测到15个差异表达的miRNA。其中9个miRNA在冬眠期间显著下调（miR10b-3p、miR-136-3p、miR-181c-5p、miR-200a-3p、miR-200b-5p、miR-200c-3p、miR-320b、miR-320c及miR-320d），6个miRNA显著上调（miR-15a-5p、miR-15b-3p、miR-15b-5p、miR-16-5p、miR-92a-3p、miR-150-5p）。另有12个miRNA未发现可识别的靶基因，但miR-200a-3p、miR-200b-5p与miR-200c-3p被证实为SERPINC1的靶基因，该基因负责编码抗凝血酶（AT）。 结论：冬眠黑熊体内存在12个差异表达的miRNA。尤为关键的是，miR-200a-3p、miR-200b-5p及miR-200c-3p在冬眠期间均呈下调状态，且与SERPINC1的表达上调及抗凝血酶的生成相关。抗凝血酶是一种强效抗凝物质，这一发现或可解释冬眠黑熊如何实现自身抗凝并免受VTE侵袭。本研究旨在比较冬眠黑熊与夏季活跃状态同类的血浆miRNA表达差异。需说明的是，由于部分黑熊在夏季采样时无法获取同一只个体的样本，因此差异表达分析未采用配对设计。