Comprehensive microRNA profiling in B-cells of human centenarians by massively parallel sequencing
© Gombar et al.; licensee BioMed Central Ltd. 2012
Received: 6 February 2012
Accepted: 16 July 2012
Published: 31 July 2012
MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and play a critical role in development, homeostasis, and disease. Despite their demonstrated roles in age-associated pathologies, little is known about the role of miRNAs in human aging and longevity.
We employed massively parallel sequencing technology to identify miRNAs expressed in B-cells from Ashkenazi Jewish centenarians, i.e., those living to a hundred and a human model of exceptional longevity, and younger controls without a family history of longevity. With data from 26.7 million reads comprising 9.4 × 108 bp from 3 centenarian and 3 control individuals, we discovered a total of 276 known miRNAs and 8 unknown miRNAs ranging several orders of magnitude in expression levels, a typical characteristics of saturated miRNA-sequencing. A total of 22 miRNAs were found to be significantly upregulated, with only 2 miRNAs downregulated, in centenarians as compared to controls. Gene Ontology analysis of the predicted and validated targets of the 24 differentially expressed miRNAs indicated enrichment of functional pathways involved in cell metabolism, cell cycle, cell signaling, and cell differentiation. A cross sectional expression analysis of the differentially expressed miRNAs in B-cells from Ashkenazi Jewish individuals between the 50th and 100th years of age indicated that expression levels of miR-363* declined significantly with age. Centenarians, however, maintained the youthful expression level. This result suggests that miR-363* may be a candidate longevity-associated miRNA.
Our comprehensive miRNA data provide a resource for further studies to identify genetic pathways associated with aging and longevity in humans.
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Mature miRNAs, between 18–25 bp in length, are transcribed as primary-miRNA (pri-miRNA) molecules which contain a characteristic stem loop structure. This stem loop targets pri-miRNA for processing by a number of RNAses, namely Drosha and Dicer, which produce a short RNA duplex. From the duplex one or both strands is incorporated into the RNA inducing silencing complex (RISC), resulting in an active miRNA. Active miRNAs target the 3′ UTR of a mRNA based on sequence homology. The nucleotides in the 2–7 position of the 5′ end of the mature miRNA comprise a “seed region”. Once an mRNA is targeted by a miRNA it can be degraded or its translation can be repressed through conserved mechanisms leading to downregulation of gene expression[1, 3].
First described in C. elegans, many miRNAs have been discovered across a wide range of species, including humans. There is increasing evidence that miRNAs are critical in a number of essential biological processes, including cell differentiation, immune response, cancer[6, 7], and life span. Thus far, 1048 human miRNA sequences have been identified through cloning, sequencing, or computational analysis (mirBase, release 16, 2010)[9–11].
The multitude of important roles played by miRNAs suggests that they are a critical component of gene regulatory networks. However, the quantification of miRNAs has been technically challenging due to their small size, low copy number, interference from other small RNAs, and contamination by degradation products of mRNAs or other RNA species. Until recently only known and computationally predicted miRNAs have been interrogated using hybridization-based array methods, which suffer from cross-hybridization, and inability to discover novel miRNAs due to limited array content. The increased availability and affordability of massively parallel sequencing now offer an opportunity to gain a high-resolution view of miRNA expression, overcoming past experimental limitations. MiRNA-seq has been utilized to discover novel and quantify expression levels of miRNAs in several species, including humans[13, 14].
Expression levels of genes are heritable in humans as quantitative phenotypes, measurable in a variety of cell types, including B-cells. Recent studies, including our own[16, 17], have demonstrated that B cells reflect functional characteristics of the donor and can be a useful tool for studying genotype-driven molecular endpoints such as gene expression, and expression quantitative trait locus (eQTL) analysis[18, 19]. B cells can act as surrogate tissues whenever there is correlation between the expression levels of B cells and phenotypes of interest[20, 21] and a large number of eQTLs originally identified in B cells can also be detected in multiple primary tissues[22, 23]. Thus B-cells have been increasingly utilized for expression quantitative trait loci (eQTLs) studies[19, 24] and as a cell model to assess gene expression responses. While comprehensive mRNA expression data for human B-cells, obtained by RNA-seq, are available for this purpose, to date no such analysis was performed to identify miRNAs expressed in B-cells. Here, we present comprehensive miRNA transcriptome profiles of B-cells from Ashkenazi Jewish centenarians and younger control individuals by miRNA-seq, providing a resource that could serve as a basis for establishing gene regulatory interactions between miRNAs and their target mRNAs in human B-cells.
Discovery of miRNAs expressed in B-cells of centenarians and controls
Novel miRNAs discovered in B-cells, their chromosome location, mature sequence, and putative folding structure
chr11:95714237–95714346 || chr11:41380469- 41380478
chr15:81221792–81221896 || chr15:545116- 545175
chr11:121147952–121148061 || chr11:13287835- 13287776
IsomiRs and variability of miRNA processing
Representative IsomiRs detected in B-cells
MiRNAs differentially expressed between centenarians and controls
Differentially expressed miRNAs in B-cells from centenarians vs. controls
Up regulated in
Control counts (standard error)
Centenarian counts (standard error)
Bonferonni adjusted p values
Cross platform comparison of differential miRNA expression
Targets of differentially expressed miRNAs
Representative GO categories enriched in targets of differentially expressed miRNAs
p-Value (Bonferonni Adjusted)
1.52 × 10-9
cellular metabolic process
3.82 × 10-6
1.05 × 10-5
6.66 × 10-5
8.02 × 10-4
4.5 × 10-2
Cross-sectional analysis of miRNA expression in different age groups
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 × 108 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[13, 31]. 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.
Relevance of miRNAs differentially expressed in centenarians to senescence and aging
Up regulated in centenarians
Down regulated in centenarians
Up regulated in senescence*
Down regulated in senescence*
Down regulated in aged PBMC**
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.
Longevity is known to have a genetic component in humans. While the heritability of average life expectancy has been estimated to be only ~25%[55, 56], 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[16, 59].
Recently, miRNAs have emerged as critical regulators of gene expression and a link between multiple miRNAs and longevity[8, 60] 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[61, 62], 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* (Figure4). 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[15, 63]. B cells act as surrogate tissues whenever there is a correlation between expression levels in these cells and phenotypes of interest[20, 21]. 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.
Massively parallel sequencing technology allowed us to accurately detect miRNAs expressed in B-cells. Considering the increasing use of B-cells for genetic and functional studies[16, 64] our data provides a resource for designing gene expression studies and to study gene regulatory networks mediated by miRNAs. Furthermore, our results from B-cells established from a human longevity cohort may generate an opportunity to explore the possible role of miRNAs in human aging and longevity and to identify genes and pathways that are targets for age-related alteration.
Population and sample collection
All individuals are enrolled in the Longevity Genes Project, and were recruited as described previously. Informed written consent was obtained in accordance with the policy of the Committee on Clinical Investigations of the Albert Einstein College of Medicine. All blood samples were processed at the General Clinical Research Center at the Albert Einstein College of Medicine in order to produce EBV transformed immortalized B-cells as a source of RNA. Total RNA was extracted from immortalized B-cell lines established from centenarians and controls using TRIZOL reagent as recommended by the manufacturer (Invitrogen).
Experimental research reported in this manuscript has been performed with the approval of the Committee on Clinical Investigations of the Albert Einstein College of Medicine. Research carried out in this manuscript is in compliance with the Helsinki Declaration (http://www.wma.net/e/policy/b3.htm).
MiRNA-seq was performed as recommended by the manufacturer (Illumina small RNA prep kit v 1.5). 10 μg of total RNA from each sample was resolved on a 15% TBE-Urea polyacrylamide gel followed by the excision of gels corresponding to the 17–35 nucleotides. Small RNAs were isolated from the gel in 300 μl of 0.3 M NaCl for 4 hours at room temperature. The small RNAs were ligated with a biotinylated RNA-DNA 3′-adaptor, gel-purified, and ligated with a 5′-adaptor. Products with both adaptors were gel-purified, reverse-transcribed, and PCR amplified for 14 cycles. Sequencing was performed on an Illumina GA1 analyzer.
The sequencing data was provided from the GA1 sequencer in a standard fastq format. The fastq files were trimmed of adapter sequences and of low quality reads (reads which had more than 3 base-calls below sufficient quality value), through a c++ program. These sequences were then collapsed to remove redundancy using the Galaxy Genome Browser tool fastx. At this point sequences from each of the samples was aligned to the known human miRNA/small RNA database or put into the mirDeep pipeline for the discovery of novel miRNAs. To normalize the samples we determined how frequently the miRNA was annotated per the number of reads reported from the sequencer. The basis of this idea is that for a given number of small sequences isolated from a cell there should be on average the same number of total miRNAs from the sample. Read counts from different libraries were normalized to the total reads in each sample.
After normalization any reads observed in fewer than 3 samples or with a copy number less than 10 were not considered for analysis; this correction removed extremely low abundance miRNAs. To identify differentially expressed miRNAs, the data was analyzed through Fisher’s exact test using a Bonferonni correction[33–35] for multiple hypothesis testing. Those miRNAs meeting a corrected cutoff with a p-value below 0.05 and with a fold change greater than 2.0 were considered differentially expressed.
Quantitative RT- PCR analysis
Total RNA was isolated from B-cells using RNA isolation kit (Qiagen, Valencia, CA) and then converted to complementary DNA using TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA) with microRNA specific RT primer (Applied Biosystems). A TaqMan® microRNA assay was performed using AB StepOneTM real-time PCR system to quantify relative miRNAs expression in these samples. The 20 μl total volume final reaction mixture consisted of 1 μl of TaqMan microRNA specific primer, 10 μl of 2x Universal Master Mix with no AmpErase® UNG (Applied Biosystems) and 1.3 μl of complementary DNA. PCR was performed using the following conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, and 60°C for 1 min. All reactions were run in duplicate to reduce confounding variance. U6 snRNA (Applied Biosystems) was used as an internal control. Means from different conditions were compared using the Student’s t-test. A significance threshold of P < 0.05 was used.
The sequence data from this study have been submitted to the submitted to the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE32493 All novel microRNA sequences have been submitted to miRBase (http://www.mirbase.org).
This work has been supported by grants to Dr. Y. Suh from the Glenn Award for Research in Biological Mechanisms of Aging, from the US National Institute of Health (RO1 AG024391, PO1 AG027734, and PO1 AG17242), and from a pilot grant from the Einstein Cancer Center.
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