The naked mole-rat is not only the longest-lived rodent, but has a much longer lifespan than expected for its relatively small body size and has been shown to be extremely resistant to neoplasia. Furthermore, since a number of other rodents including mice, rats and guinea pigs already have had their genome sequenced, the naked mole-rat is a prime candidate for comparative genomics studies. In this study, we compared the expression of genes between naked mole-rats and mice by directly sequencing cDNA libraries constructed from liver samples with the aim to gather insights regarding the molecular mechanisms responsible for the naked mole-rat adaptations as well as develop methods for functional genomics in this exotic species.
Differential expression analyses using 2nd
-generation sequencing platforms such as Illumina/Solexa, ABI SOLiD have increased in popularity due to their low cost and high throughput 
. However, due to the short lengths of their read output, a reference genome is also needed and has limited the number of 2nd
-generation transcriptome studies in non-sequenced species. In our study, we show that it is possible to conduct differential gene expression analyses in non-sequenced mammalian species using only 2nd
-generation sequencing technologies by assembling transcriptome contigs of the non-sequenced species. Using a combination of 2nd
-generation sequencing platforms (Roche 454 and Illumina/Solexa), we were able to compare gene expression between wild-derived mice and naked mole-rats without using a naked mole-rat reference genome. Our approach and methods not employing a reference genome may be applicable to other studies of non-traditional mammalian model organisms.
One caveat of our method is that only genes over-expressed in the naked mole-rat when compared to mice can be accurately identified. Genes detected to be under-expressed in the naked mole-rat by our approach may be real biological phenomena or may be due to a lack of coverage from our assembly of the naked mole-rat transcriptome. Under-expressed ageing-related genes (with over 1,000 reads in the naked mole-rat and hence with some coverage) that caught our attention and may be subject of further studies included Gpx1
). Another study recently found lower levels of Gpx1
in naked mole-rat liver than in mouse liver 
, so some of our under-expressed genes may be phenotypically relevant. Due to the incomplete naked mole-rat transcriptome and annotations, however, it may be that Solexa read counts for some transcripts were extremely low due to poor mapping, rather than low expression. This may also explain why higher relative abundances of Hprt1
were observed in the qPCR analysis when compared to the Solexa data. Nonetheless, our analyses show that, unlike in mice, naked mole-rat A2m
, and Sat2
are expressed in the liver and demonstrate the capacity of our method to detect over-expressed genes.
We found a considerable number of genes over-expressed in the naked mole-rat, many with large expression changes. One possibility is that a shift in tissue-specificity may account for some of the dramatic over-expression levels we found so that a gene with negligible expression in the mouse liver, but some expression in the naked mole-rat's, will be detected as considerably over-expressed in the naked mole-rat liver. Another other hypothesis is that some genes evolved different functions in the naked mole-rat liver.
Candidate over-expressed genes in the naked mole-rat associated with its adaptations
In addition to longevity, mice and naked mole-rats diverge on many other physiological traits and live in extremely different natural habitats. It is therefore impossible to tell for certain whether an over-expressed gene or pathway in the naked mole-rat is responsible for its increased longevity or other phenotypic traits well suited to a subterranean milieu. In spite of this caveat, we identified genes and pathways over-expressed in the naked mole-rat that have been previously associated with ageing and therefore may provide clues for understanding the differences in longevity between these species. These may also be candidates for further functional studies. One of these genes is stearoyl-coenzyme A desaturase (Scd3) which has been detected as the 11th most over-expressed gene in the naked mole-rat (14,613 76 bp reads vs 11; 2961 39 bp reads vs 7). Scd3 is a fatty acid desaturase involved in lipid biosynthetic, lipid metabolic and oxidation-reduction processes which, as discussed later, all have previously been associated with ageing.
One of the most interesting over-expressed genes in the naked mole-rat is the serum pan-protease inhibitor, alpha2-macroglobulin (A2m). While A2m is expressed at a low level in wild-derived mouse liver (1,307 reads out of 8.8 M), we detected almost 202,500 (out of 9.0 M) reads in naked mole-rat liver which makes it among the top 20 most over-expressed genes. Interestingly, A2m is listed as a candidate protein relevant to the human ageing process in GenAge, a database of ageing- and longevity-associated genes 
. A2m is also known to interact with longevity-associated ApoE (apolipoprotein E) and is associated with Alzheimer's disease 
. Furthermore, A2m was determined to be a biomarker for ageing in vivo as its mRNA expression level showed positive correlation with age 
. The function of A2m as a proteinase inhibitor may also be of interest in the context of protein turnover regulation in naked mole-rats, which Pérez et al. (2009) argue to be a key contributor to their extreme longevity.
Another gene that caught our attention is Epcam
, also known as Tacstd1
, which is at the top of our list with 37,073 76 bp reads in NMR and only 27 in mouse (596 39 bp NMR vs 4 mouse reads). Epcam is a transmembrane glycoprotein hypothesized to function as a cell adhesion molecule which interferes with cadherin-mediated cell–cell contact 
. Furthermore, Epcam has been shown to promote cell cycling and enhance proliferation 
and it is found to be over-expressed on epithelial progenitors, carcinomas and cancer-initiating cells 
. While we cannot explain the over-expression of a tumour-associated gene in the cancer-resistant naked mole-rat, we suggest Epcam is a promising target for future studies. In the context of cancer, another strongly over-expressed gene (3rd
in our list) in the naked mole-rat of potential interest is Gulp1
, an adaptor protein that promotes phagocytosis of apoptotic cells 
, and might therefore contribute to species differences in tissue turnover. Lastly, one gene recently hypothesized to be important in species longevity is Nrf2 
, also known as Nfe2l2, which we interestingly found over 6-fold over-expressed in the naked mole-rat (Dataset S1
). All in all, our findings warrant further studies of a number of genes (our full results are available in Datasets S1
) and open new research avenues.
Candidate pathways for extreme longevity divergence in the naked mole-rat
Within over-expressed genes in the naked mole-rat, genes associated with oxidoreduction were strongly overrepresented as well as genes associated with mitochondria and more specifically mitochondrial matrix. Consistent with the free radical theory of ageing, the over-expression of genes related to oxidoreduction could protect the naked mole-rat from reactive oxygen species. Indeed, the succinate dehydrogenase cytochrome b560 subunit (Sdhc), a member of the mitochondrial electron transport chain, was one of the few ageing-related genes strongly over-expressed in the naked mole-rat. However, the naked mole-rat exhibits greater levels of oxidative damage in lipids, DNA and proteins 
, thus the over-expression of the oxidoreduction and mitochondria genes could reflect a higher need for ROS protection and/or the cause of those higher levels. Given that naked mole-rats have a lower body temperature than mice 
, in addition to living in the aforementioned hypoxic environment, differential expression of genes related to metabolism and energy may be due to other physiological differences between these species. Nonetheless, in view of the hypothesis that mitochondria play a major role in mammalian ageing 
, these results point towards a putative role for oxidoreduction and mitochondrial alterations in the long lifespan of the naked mole-rat.
Although borderline significant, it is noteworthy that both fatty acid metabolism and lipid biosynthetic process functional groups figured among the top categories of over-expressed genes in naked mole-rats. According to Hulbert (2008), membrane fatty acid composition is correlated with the maximal lifespans of mammals through the reduction of oxidative damage caused by the autocatalytic ROS products of lipooxidation 
. Another borderline significant category among over-expressed genes was endoplasmic reticulum, in which alterations have been suggested in naked mole-rat cells 
In conclusion, the largest effects on gene expression in comparing naked mole-rat and mouse liver RNAs suggest hypotheses about redox testable by genome engineering in the mouse. By employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing, our differential gene expression analysis obviated the need for a reference naked mole-rat genome and paves the way for gene expression profiling of the naked mole-rat. Nonetheless, further studies of Heterocephalus glaber
, an emerging model of successful ageing and cancer resistance, would greatly benefit from a reference genome sequence and we are currently developing efforts in this direction (http://www.naked-mole-rat.org/