We employed a massively parallel sequencing technology to identify miRNAs expressed in B-cells established from Ashkenazi Jewish Centenarians and younger elderly controls without a family history of longevity. In this study, which represents the first comprehensive studies to analyze miRNAs in human B-cells, we obtained 26.7 million reads comprising 9.4
bp from 3 centenarian and 3 control individuals. We found a total of 284 miRNAs expressed in human B-cells, eight of which were previously unknown, putative novel miRNAs.
Profiling miRNA transcriptomes has gained importance with increasing evidence for the role of miRNA expression in defining cellular phenotypes. Prior to the advent of increasingly cost-effective, small RNA sequencing, microarrays were the prevailing methods for miRNA expression analysis. However, apart from noise due to cross-hybridization to probes with similar short-sequences, array technologies are limited in several key ways. First, microarrays are limited to the probe sets, eliminating the possibility to discover novel miRNAs. Second, arrays are only able to identify the relative abundance of miRNAs but not their absolute numbers
]. Sequencing allows accurate detection of expression levels over a wide dynamic range, including low copy number miRNAs and subtle fold changes between test and control groups. Finally, array-based methods cannot differentiate isomiRs (mature miRNAs originated from the same precursor but differing in one or more bases) resulting from the high variability in miRNA processing
]. Using miRNA-seq of human B-cells, we were able to identify novel miRNAs and isomiRs, and detected expression of low-copy miRNAs and subtle fold changes between centenarians and controls, which could not be measured using TaqMan qPCR.
Genome-wide transcriptional profiles of miRNAs in C. elegans
] and human blood cells
] showed that the majority of miRNAs decreased in expression with age. Likewise, cellular senescence was accompanied by significant down-regulation in miRNA expression
]. Our results suggest that centenarians may be more resistant to such age-related down-regulation of miRNAs because the overwhelming majority of significant miRNAs are increased in centenarians as compared to controls in B-cells: we found 22 miRNAs significantly upregulated, with only 2 downregulated, in centenarians as compared to younger controls. Strikingly, expression levels of 5 miRNAs shown to be increased in centenarians (hsa-miR-148a, hsa-miR-345, hsa-miR-361-5p, hsa-miR-192, hsa-miR-454) have been demonstrated to be down-regulated during cellular senescence (Table
). Among these, hsa-miR-148a was also found to be down-regulated with age in peripheral blood mononuclear cells (PBMCs)
]. Together, these results suggest that miRNAs associated with longevity can be detected by cross-sectional expression analysis as utilized in this work.
Relevance of miRNAs differentially expressed in centenarians to senescence and aging
By conducting a cross-sectional analysis of miRNA expression in different age groups, we found that expression of hsa-miR-363* significantly declined with age in control individuals, whereas centenarians maintain comparably high expression levels, similar to the observed in the middle age group. The results suggest that hsa-miR-363* is a candidate longevity-associated miRNA. Previously miRNA* sequences were thought to be degraded. However a growing body of work challenges the dogma that miRNA* is simply a non-functional byproduct of miRNA biogenesis, suggesting instead that miRNA* plays a significant role in cellular function and human disease
]. Furthermore, they are implicated in the aging of C. elegans
]. The predicted targets of hsa-miR-363* include PTEN, BCL2, AKT1, and IGFBP5 among genes listed in the GenAge database
]. To our knowledge this is the first cross-sectional analysis of miRNA in a human longevity cohort, identifying a potential longevity-associated miRNA.
Targets for our differentially expressed miRNAs were obtained through the literature and by bioinformatics means (Additional file
: Table S4). The predicted and validated targets of the 24 differentially expressed miRNAs are enriched in biological pathways implicated in the aging process, including cell differentiation, cell cycle, cell signaling, and cell metabolism (Table
), as well as the GenAge database
]. Of particular interest were the genes involved in cognitive aging (ADAMTS5, APPL1, IGF2, LRP1b), aging-related signaling/transcription (E2F2, FGF7, MLL, MYB , KLF3, PTEN, RICTOR, SMAD2, SOCS5, TCF4), cell cycle/senescence/apoptosis (BCL2L11, CBL , CDC2L6, E2F2, EEF2K), and overlap with the GenAge database of known aging related genes (SHC1, E2F1, STAT3, IGF1, IRS2, PTEN, RPA1, BCL2, SP1, HBP1, MXD1). We searched for potential target genes that were predicted to bind multiple miRNAs found to be differentially expressed in our study because it has been shown that miRNAs can act in an additive manner and the co-targeting by multiple miRNAs would greatly reduce mRNA expression even if the miRNAs were in low copy number
]. We found that RICTOR (rapamycin-insenstive companion of mTOR) was predicted to bind at least two miRNAs upregulated in centenarians as compared to controls. The 3′
UTR of RICTOR and the binding sites for miR-148a and miR-155 (fold change
1.5) are shown in Figure
. RICTOR is part of the TOR family of genes which are integral to growth and proliferation, and down-regulation of this pathway is shown to extend lifespan
3′UTR of Rictor showing multiple miRNA target locations.
Longevity is known to have a genetic component in humans. While the heritability of average life expectancy has been estimated to be only ~25%
], studies of centenarians indicate much stronger heritability at old age. For example, siblings of centenarians have a 4 times greater probability of surviving to age 90 than siblings of people with average life span
]. Achieving a lifespan of 100 years is 17 and 8 times more likely for male or female siblings of centenarians, respectively, compared to their birth cohort
]. These findings firmly established the utility of human centenarians as a model system to unravel the genetics of longevity. For this study, we used our unique cohort of centenarians and elderly controls without family history of exceptional longevity, all of genetically homogeneous Ashkenazi Jewish descent. This cohort has been used to successfully discover longevity associated genotypes and phenotypes in the past
Recently, miRNAs have emerged as critical regulators of gene expression and a link between multiple miRNAs and longevity
] and aging
] has been demonstrated in C. elegans
, implicating their role in regulation of lifespan and in the aging process. Since a significant number of miRNAs are evolutionarily conserved
], it is conceivable that miRNAs play a role in human longevity as well. Our finding that most of the differentially expressed miRNAs were upregulated in centenarians could point towards increased resilience of centenarians against an age-related decline in gene regulatory control. By conducting a cross-sectional expression analysis, we found a candidate longevity-associated miRNA, hsa-miR-363* (Figure
). Hsa-miR-363* shows reduced expression in control individuals with advancing age while maintaining relatively high expression levels in centenarians. Maintenance of youthful expression patterns may be beneficial and longevity-associated miRNAs may confer robustness to gene expression networks, protecting them against age-related deterioration.
In the field of human genetics, most of the surveys of gene expression have been conducted in B-cells because they are readily available and can be used multiple times under controlled experimental conditions
]. B cells act as surrogate tissues whenever there is a correlation between expression levels in these cells and phenotypes of interest
]. However, caution needs to be taken in interpreting the results, especially with negative data, as truly tissue-specific genes will not be detected in B-cells. The lack of a correlation can never be used to infer that the miRNA/gene is not involved in human longevity, and only positive results should be interpreted as in most large-scale discovery-based science.