Blood miRNomes and transcriptomes reveal novel longevity mechanisms in the long-lived bat, Myotis myotis

Of all mammals, bats possess some of the most unique and peculiar adaptations that render them as excellent models to investigate the mechanisms of extended longevity and potentially halted senescence. They are considered the ‘Methusalehs’¹ among mammals due to their exceptional and surprising longevity given their body size and metabolic rate [1, 2]. Typically mammals that are small have a high metabolic rate (e.g. shrews) and do not live for a long time [1, 3]. However, despite their small size and high metabolic rate bats can live for an exceptionally long time, with the oldest recorded bat (wild caught as an adult) ever recaptured being >41 years old (Myotis brandti, 7 g, Brandt’s bat) [2, 4, 5]. Indeed, to get a positive correlation between longevity and body size in mammals, bats must be removed from the analyses [1]. By comparing the ratio of expected longevity to that predicted from the ‘non-bat placental mammal’ regression line (longevity quotient- LQ) only 19 species of mammals are longer lived than man, one of these species being the naked mole rat and the other 18 are bats [1]. This suggests that bats have some underlying mechanisms that may explain their exceptional longevity.

MicroRNA (miRNA) are a subset of short (~22 bp) endogenous non-coding RNA that play a significant role in post-transcriptional regulation, via repression of translation. Typically, the 5′ end of a mature miRNA (seed region) predominantly determines the specificity and sensitivity in binding the target mRNA through either perfect or partial complementarity to the mRNA 3′-UTR, resulting in mRNA degradation or translational repression [6]. Since the first miRNA, lin-4, was discovered in 1993 [7], a multitude of miRNA have subsequently been identified, and implicated in the regulation of the vast majority of biological pathways including cell cycle regulation, metabolism, tumorigenesis, as well as immune response. However, the role of miRNA regulation in mammalian ageing and the onset of age-related diseases has only recently been established [8].

In mammals, various miRNA have been shown to be differentially expressed during ageing, most of which appear to be generally tissue-specific [8]. It was reported that the expression of miR-93 remarkably increased in liver tissues from extremely old mice and rats [9, 10]. miR-93 targets glutathione-S-transferases (GST) and SIRT1 genes [9, 10], which are vitally important in oxidative stress defense and metabolic homeostasis. Another example is miR-144. It is significantly up-regulated in aged human brains, which targets ataxin-1, the gene associated with spinocerebellar ataxin type 1 (SCA1) [11]. In addition to tissue-specific ageing, it is increasingly evident that many miRNA regulate gene expressions in well-known ageing pathways, most notably in the p53 tumor suppressor pathway (miR-34, miR-29 and miR-217, etc.) and insulin-like growth factor signaling pathway (IIS) (miR-17-92 family and miR-214, etc.) [8]. As altered patterns of miRNA expression can contribute to mammalian ageing, an investigation of the genome-wide miRNA profile (miRNome) may help elucidate the molecular mechanisms behind the extreme longevity of bats.

Despite being the second largest order of mammals (~1200 species) [12], there is a scarcity of genomic and transcriptomic bat resources. To date, only five well-annotated bat genomes (Myotis lucifugus, Myotis brandtii, Myotis davidii, Pteropus alecto and Pteropus vampyrus) are publically available. Phylogenomic studies of bat genomes and other mammalian species reveal that a number of genes are under positive selection in bats [5, 13, 14]. These genic adaptations have been correlated with traits such as echolocation, powered flight, hibernation, immunity and longevity. For example, specific non-synonymous mutations in GHR and IGF1R, key ageing-related genes, were detected in several long-lived vespertilionid bats (M. brandtii, M. lucifugus and Eptesicus fuscus) [5], while a large proportion of genes involved in DNA repair (RAD50, KU80, MDM2 etc.) and the NF-кB pathway (c-REL and ATM2 etc.) were reported to be under positive or divergent selection in M. davidii and P. alecto [14]. These results suggest bats may better detect and repair DNA damage. Intriguingly, positive selection was also detected in mitochondrial-encoded and nuclear-encoded oxidative phosphorylation genes in bats, which may explain their efficient energy metabolism necessary for flight [15]. Apart from comparative genome analysis, only a small number of transcriptomic studies on bats using mRNA-Seq and miRNA-Seq technologies have been carried out, focused primarily on the characteristics of hibernation [16], immunity [17, 18], echolocation [19] and phylogeny [20]. However, the molecular mechanisms of adaptations affecting longevity are still far from understood, especially with respect to gene regulation.

In the present study, we sequenced six small RNA libraries from whole blood sampled from wild-caught greater mouse-eared bats (Myotis myotis) and for the first time made genome-wide comparisons of both miRNomes and mRNA transcriptomes between bat and non-bat mammalian species (human, pig and cow). Whole blood is a nonlethal sample type that can reflect the real time overall physiological status of an individual [21]. Our earlier study showed that only a few drops of whole blood (<200 μl) contains a high percentage of genome-wide and body-wide transcripts [22]. We then compared both the corresponding bat blood miRNome and transcriptome to those of non-bat mammals (human, pig, cow), to elucidate bat-specific gene expression as well as post-transcriptional regulation.

The profiling of the M. myotis blood miRNome showed a large number of bat-specific miRNA involved in regulating important pathways related to immunity, tumorigenesis and ageing. Comparative analyses of both miRNomes and transcriptomes also revealed distinctive longevity mechanisms in bats. Several up-regulated miRNA possibly act as tumor suppressors. Gene Ontology (GO) enrichment analysis of differentially expressed protein-coding genes showed that up-regulated genes in bats compared to other mammals were mainly involved in mitotic cell cycle and DNA damage repair pathways while a high number of down-regulated genes were enriched in mitochondrial metabolism. The results and data presented here show unique regulatory mechanisms for protection against tumorigenesis, reduced oxidative stress, and robust DNA repair systems, likely contribute to the extraordinary longevity of bats.

Mining miRNA from deep sequencing reads is highly dependent on the genome, which enables the prediction of their compact pre-miRNA hairpin structures. In this study, we employed the M. lucifigus genome as a proxy reference since the M. myotis genome has not yet been published. Although suffering some losses in the number, most abundant and conserved miRNA can be identified in a closely related species’ genome [23]. This is also shown by large number of miRNA genes (539) found in this study using the strict pipeline, the number of which falls within the range observed for other bat studies using miRNA-Seq, although different tissues and sequencing depths were used [17, 20]. The distribution of miRNA genes displayed no particular bias for genomic location. However, the largest category of miRNA (39.8 %) was in the intergenic regions (Fig. 2c), which is similar to human (49.9 %) and mouse (44.8 %) [24]. A large number of intragenic miRNA, which were located within introns and exons, were also found, and it is hypothesized that they may co-express with their host genes because of shared promoter sequences [25, 26]. Interestingly, we also detected 18 miRNA genes spanning the boundaries of exons and introns (Fig. 2c). These findings suggest the complexity of transcriptional splicing activity, and that the locations of miRNA can affect their expression and function. In addition, we identified and analyzed the paralogous groups in the M. myotis blood miRNome, observing that a large proportion of mature miRNA (78.4 %) were single-copy miRNA (Fig. 2e). Paralogous miRNA can shed light on gene duplication events, evolutionary history and post-transcriptional RNA editing. However, few studies on mammalian miRNA paralogs are available for comparison and analysis, probably due to the poor annotation of noncoding regions of mammalian genomes.

A significant proportion of miRNA (Fig. 2d) had no homologs in other species, reflected by many novel seed regions and bat-specific miRNA identified in this study. Potentially this could result from a lack of taxon-wide miRNA studies based on well-assembled and annotated genomes. However, a recent study estimated that the proportion of miRNA orthologous pairs between human and mouse was only 16 %, whose genomes are better annotated, leaving a large abundance of species-specific miRNA [24]. miRNA experience a rapid birth and death rate across species, and species-specific miRNA are considered to be evolutionarily recent. Not surprisingly, the expression level of these novel miRNA tended to be significantly lower (P < 0.05) than that of conserved miRNA (Fig. 3a). Abundantly expressed miRNA are highly likely to be functionally important, evolutionarily conserved, and under purifying selection; while novel miRNA with low expression level may have a comparatively weaker effect on their potential target transcripts, yet could convey species-specific adaptations. Occasionally, miRNA with low expression may be selectively favored and will be maintained in the genome. A direct correlation between the numbers of miRNA and biological complexity has been proposed, suggesting that miRNA innovation may play a key role in the emergence of increasingly novel and complex traits [27]. To understand the function of the bat-specific miRNA, we constructed the miRNA-mRNA network and performed function enrichment analysis on their targets. Many of the pathways and functions were associated with metabolism, immunity and protein homeostasis (Fig. 3b). Simultaneously, analyses of species-specific miRNA from other mammals (pig, cow and human) revealed common GO terms shared with bat, suggesting different, convergent species-specific miRNA can target the same pathways. The bat-specific GO terms, however, indicated the acquisition of novel miRNA in bats which regulate ageing and cellular senescence.

For bat, the most representative enriched terms, which are highly related to ageing and cell senescence, were ‘aging’ (GO: 0007568) and ‘guanyl-nucleatide exchange factor activity’ (GO: 0005085). Remarkably, some cell cycle mediators which accelerate cell growth and proliferation were included under the term ‘aging’, such as GRB2, CASP7, SHIP1 and GSN. It is commonly accepted that accelerated cell growth directly coincides with tumorigenesis and cancer progression. Gelsolin (GSN), which functions as a cell growth promotor and apoptosis inhibitor, has been implicated in the oncogenesis of certain cancers [28]. Previous study showed its overexpression promoted the proliferation and mobility of hepatocellular carcinoma (HCC) cells [28]. Another example is GRB2, an activator of Ras/Raf/MEK/mitogen-activated protein kinase pathway (MAPK). It is reported that its downregulation could inhibit breast cancer cell growth [29]. Interestingly, we also noticed SHIP1, which limits blood cell production and immune regulatory cell numbers. The inhibition of SHIP1 was shown to trigger apoptosis of blood cancer cells, suggesting the potential target for the treatment of hematological malignancies [30]. As well as inhibition of cell growth, bats may suppress inflammation and tumorigenesis by targeting guanyl-nucleotide exchange factors (GEFs). GEFs play a fundamental role in many diverse signaling pathways related to immunity, cell growth and tumorigenesis. RasGRP1, 2, 3 and SOS2 are all GEFs which activate Ras small GTPases and are some of the 50 GEFs targeted by bat-specific miRNA predicted in this study. RasGRP1 and 3 are recruited during the signaling cascades instigated by T-cell and B-cell receptor stimulation, respectively [31]. They are essential for the activation of Ras and promoting proliferation in T and B-cells in response to infection. Unsurprisingly, these genes have been shown to be overexpressed in a myriad of cancers, including melanoma, T-cell acute lymphoblastic leukemia (T-ALL) and prostate cancer [32, 33], making inhibitors of these proteins possible therapeutics. These results imply that M. myotis are capable of regulating cell cycle regulators as well as Ras small GTPases through degradation of direct or indirect activators. This may prevent tumor or cancer development and immune-related disorders, contributing to their long lifespan.

Comparisons of conserved miRNA expressions across species can also reveal distinct adaptations. However, there are two major obstacles to compare miRNA across species. First, there were limited miRNA resources that could be used for comparison. We employed data from human, pig and cow because they had both blood miRNomes and transcriptomes sequenced from healthy adults using NGST. This makes the investigation of the correlation between miRNA and mRNA expressions possible. Second, the existence of paralogs made it difficult to accurately quantify the orthologous miRNA across species [34]. Therefore, we calculated the geometric mean to represent the expression of each paralogous group. This is because all these paralogs were under strict inspection that they shared the same seed region, suggesting a high possibility of targeting the same mRNA. Due to a large number of bat-specific miRNA identified, it is not surprising that only 37 conserved orthologous groups were found across all 18 libraries. Cluster analysis and PCA indicated the differences within biological or technical replicates were much smaller than the differences between species (Fig. 4a, b).

Six conserved miRNA were found differentially expressed between bat and non-bat species (human, pig and cow), most of which were highly likely to be biologically functional in oncogenic or immune pathways. Among the 4 up-regulated miRNA, the expression of miR-101 in bat was at least three times than that in other mammals (Fig. 5). It is reported that miR-101, which functions as a tumor suppressor, is down-regulated in several cancers. Its overexpression inhibits cell proliferation and promotes apoptosis in breast cancer cells by inhibiting Jak2 [35]. Similarly, expressions of miR-16 and miR-143 inhibit cell proliferation and suppress tumorigenesis, and miR-143 has been observed to be down-regulated in cervical cancer [36]. Importantly, one down-regulated miRNA, miR-221, was reported to function as a tumorigenesis promoter in human breast cancer. These findings demonstrate M. myotis are likely better at suppressing tumorigenesis than human, pig and cow, achieved by overexpression of miRNA associated with tumor suppression and inhibition of miRNA which promotes tumorigenesis. miR-155 is a multifunctional miRNA regulating inflammation, immunity and oncogenic pathways [37]. Interestingly, miR-155 also targets Bach1, a negative regulator of Nrf2, which is a core activator of the antioxidant response signaling pathway [38]. Activation of this pathway is a major mechanism for cells to defend against oxidative and electrophilic stresses by enhancing cellular antioxidant activity. This indicates that M. myotis efficiently degrade Bach1 mRNA and activate the Nrf2 pathway, thus increasing defenses against cellular damage caused by reactive oxygen species (ROS). These miRNA regulatory mechanisms through which M. myotis can prevent tumorigenesis, inflammatory diseases and cellular oxidative damage, may explain their incredible longevity. Interestingly, transcriptome and proteome analysis of another long-lived mammal, the naked mole rat, also indicated their resistance to tumorigenesis, and insusceptibility to oxidative damage with age [39]. This demonstrates that long-lived mammals may regulate and control common ageing pathways to reach old age.

To address the uniqueness of bats from a different perspective, we also compared the RNA-Seq transcriptomes between bat and non-bat mammals. However, it is also a challenge to compare transcriptomes across species due to the existence of many paralogs. The quantification of paralogs calculated by mapping the clean reads to the genome cannot reflect the true expression, as paralogs are highly similar so that it is impossible to tease apart the reads having multiple mapping coordinates. Therefore, we only obtained all 1:1 single-copy orthologous protein-coding genes across bat, human, pig and cow genomes for analysis. Surprisingly, we identified many more down-regulated genes than up-regulated genes in bat (Fig. 7a, b). It was a concern that the quality of bat transcriptomes might be lower than that of other species, leading to the identification of massive down-regulated genes in bat. However, this concern was obviated by a measure of transcriptome completeness (CEGMA) which showed their qualities were highly comparable (bat: 64.9 %, human: 70 %, pig: 69 % and cow: 65.7 % in completeness).

Based on the enrichment results, a large number of mitochondria-related genes were down-regulated in bat (Fig. 7c, d). Mitochondria are the powerhouse of the cell generating adenosine triphosphate (ATP) from oxygen and the products of glycolysis. However, reactive oxygen species (ROS), such as superoxides and peroxides generated by the premature reduction of oxygen, can damage DNA, RNA and proteins, contributing to the pathophysiology of ageing. It is noteworthy that many of these down-regulated genes encode proteins, such as NDUFV1 and NDUFA1, which are components of electron respiratory chain complex I, the primary site of ROS generation [40]. Reduced expression of mitochondrial-associated transcripts may represent a general mechanism where cells protect themselves against oxidative stress [41]. Our finding is consistent with the study of oxidative stress in another long-lived bat M. lucifugus, concluding that their mitochondria produce half to one-third the amount of hydrogen peroxide per unit of oxygen consumed than that of relatively short-lived mammals of similar size, short-tail shrew and white-footed mouse respectively [42].

On the other hand, Myotis bats seem to have evolved a heightened DNA repair system, showing up-regulation of several DNA repair genes. For example, UVRAG, encodes a protein which interacts with DNA repair enzymes to aid DNA double-strand break repair and plays a fundamental role in centrosomal and chromosomal stability [43]. Interestingly, UVRAG also interacts with Beclin 1 protein, leading to the activation of autophagy and inhibition of tumorigenesis [43]. The tumor suppressor genes BRCA1 and BRCA2, which were both up-regulated in bats, encode proteins that are pivotal in maintaining genomic stability by promoting efficient and accurate repair of DNA double-strand breaks. Mutations in these two genes will lead to the development of various types of cancers [44]. We conclude that decreased levels of oxidative stress and heightened DNA repair activity may protect bats against genomic instability, contributing to halted ageing in the long-lived M. myotis.

In summary, we established the first atlas of the blood miRNome of the long-lived bat, M. myotis, identifying a large number of miRNA involved in key ageing-related pathways. The comparative analyses of miRNomes and transcriptomes between bats and other mammals resulted in the discovery of many bat-specific miRNA, differentially expressed miRNA and mRNA, and their regulatory networks. Our results show that M. myotis exhibit increased protection against tumorigenesis and oxidative damage, heightened cellular damage repair activity and better genomic stability, all of which likely contribute to their exceptional longevity.