Let-7 microRNAs are developmentally regulated in circulating human erythroid cells
- Seung-Jae Noh†1,
- Samuel H Miller†2,
- Y Terry Lee1,
- Sung-Ho Goh1, 4,
- Francesco M Marincola2,
- David F Stroncek2,
- Christopher Reed3,
- Ena Wang2 and
- Jeffery L Miller1Email author
© Noh et al; licensee BioMed Central Ltd. 2009
Received: 12 November 2009
Accepted: 25 November 2009
Published: 25 November 2009
MicroRNAs are ~22nt-long small non-coding RNAs that negatively regulate protein expression through mRNA degradation or translational repression in eukaryotic cells. Based upon their importance in regulating development and terminal differentiation in model systems, erythrocyte microRNA profiles were examined at birth and in adults to determine if changes in their abundance coincide with the developmental phenomenon of hemoglobin switching.
Expression profiling of microRNA was performed using total RNA from four adult peripheral blood samples compared to four cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNAs were hybridized to custom spotted arrays containing 474 human microRNA species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines provided a hybridization reference for all samples to generate microRNA abundance profile for each sample.
Among 206 detected miRNAs, 79% of the microRNAs were present at equivalent levels in both cord and adult cells. By comparison, 37 microRNAs were up-regulated and 4 microRNAs were down-regulated in adult erythroid cells (fold change > 2; p < 0.01). Among the up-regulated subset, the let-7 miRNA family consistently demonstrated increased abundance in the adult samples by array-based analyses that were confirmed by quantitative PCR (4.5 to 18.4 fold increases in 6 of 8 let-7 miRNA). Profiling studies of messenger RNA (mRNA) in these cells additionally demonstrated down-regulation of ten let-7 target genes in the adult cells.
These data suggest that a consistent pattern of up-regulation among let-7 miRNA in circulating erythroid cells occurs in association with hemoglobin switching during the fetal-to-adult developmental transition in humans.
MicroRNA (miRNA) is approximately 22 nucleotide long single-stranded RNA which regulates gene expression through either post-transcriptional gene silencing by pairing to target mRNA to trigger mRNA cleavage, trafficking of mRNA for degradation, or translational repression . MicroRNAs are predicted to target over one-third of the human genome . Regulated expression of miRNA was linked to many physiological processes including developmental timing and neuronal patterning . Gene products that control a broad range of functions including proliferation, differentiation and apoptosis are targeted by miRNA [4, 5]. For example, expression of miR-145 is thought to act as a tumor suppressor in normal cells, and miR-145 is under-expressed in breast cancer. Alternatively, over-expression of a separate miRNA named miR-155 is thought to be involved in oncogenesis . Expression of some miRNA is evolutionarily-conserved including the let-7 miRNA family. Experimental findings suggest that let-7 miRNAs play major roles in growth and development . Based upon involvement of let-7 miRNA in the larval-to-adult transition in C. elegans and the juvenile-to-adult transition in Drosophila, a similar function for let-7 miRNA in mammalian development is being explored .
Birth defines the developmental transition from fetal to extra-uterine life in humans. Post-natal life necessitates the development or function of several organ systems that maintain those functions into adulthood. The loss of placental function necessitates pulmonary function and atmospheric respiration for adequate tissue oxygenation and survival of the host. Tissue oxygenation is accomplished during this developmental period via hemoglobin in erythrocytes that complete the placental or pulmonary circuits . Human hemoglobin is a heterotetrameric metalloprotein composed with four globin chains; two of alpha chains (α1, α2, ζ, μ, and θ) and two of beta chains (β, δ, G-γ, A-γ, and ε). Each globin molecule binds one heme molecule . In humans and other large mammals, the perinatal period defines a major developmental transition from fetal-to-adult hemoglobin types in erythroid cells . Hemoglobin composition switches around the time of birth from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2β2). Based upon the importance of hemoglobin switching for the clinical development of sickle cell anemia and thalassemias, this developmental hemoglobin switching process has been studied extensively. While studies of hemoglobin switching led to fundamental insights regarding gene and protein structure and regulation over the last 50 years, the molecular mechanism(s) for this developmental phenomenon remain elusive. Hemoglobin switching is accomplished via developmentally timed and coordinated changes in globin gene expression. As such, efforts remain focused upon understanding transcription regulation in erythroid cells. Since miRNA represent a new class of transcription regulators in eukaryotic cells, human circulating erythroid cells were used to determine whether fetal-to-adult hemoglobin switching is associated with changes in miRNA abundance patterns.
Preparation of reticulocyte RNA
Studies involving human subjects were approved by the institutional review boards of the National Institute of Diabetes, Digestive, and Kidney Diseases or the National Naval Medical Center. After written informed consent was obtained, peripheral blood or umbilical cord blood was collected from four adult healthy volunteers and four pregnant females. Reticulocyte-enriched pool was obtained by removing plasma, platelets, and white blood cells by centrifugation and filtering as described previously . Total RNA was isolated from the reticulocyte-enriched pool using TRIzol reagent.
Transcriptome profiling of reticulocytes from cord and adult bloods
Profiles of mRNA expression were analyzed based on total RNA from six cord blood and six adult blood samples using GeneChip® Human Genome U133 Plus 2.0 arrays (Affymetrix) with the same method as previously described .
MicroRNA array analysis
Custom spotted miRNA array V4P4 containing duplicated 713 human, mammalian and viral mature antisense microRNA species (miRBase: http://www.mirbase.org/, version 9.1) plus 2 internal controls with 7 serial dilutions was printed in house (Immunogenetics Laboratory, Department of Transfusion Medicine, Clinical Center, National Institutes of Health). Validation of this platform according to sample input, dye reversal, and labeling method efficiency were optimized for analyses of microRNA species in hematopoietic cells as reported previously . The oligo probes were 5' amine modified and immobilized in duplicate on CodeLink activated slides (GE Healthcare, Piscataway, NJ) via covalent binding. Fluorescent labeled miRNA from total RNA samples was synthesized using miRCURY LNA microRNA Power labeling kit (Exiqon, Woburn, MA) according to manufacturer's protocol. Purified total RNA from four cord blood and four adult RBC was labeled with fluorescent Hy5-dye. Reference total RNA isolated from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines were labeled with fluorescent Hy3-dye for comparison. Labeled RNA from sample and reference were co-hybridized to miRNA array at room temperature overnight. After washing, raw intensity data were obtained by scanning the chips with GenePix scanner Pro 4.0 and were normalized by median over entire array. Differentially expressed miRNAs were defined by two-tailed unpaired t-test comparing cord blood group with adult blood group as miRNAs with p-value less than 0.01 and fold change greater than two. All microarray data compiled for this study is MIAME compliant and the raw data has been deposited in a MIAME compliant database (GEO#: GSE17639, GSE17405).
Quantitative real-time PCR
To confirm the microarray results, quantitative real-time PCR (qPCR) was performed on let-7a through let-7i miRNA members in adult blood vs. cord blood. Complementary DNA specific to each miRNA was generated from total RNA using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer's protocol and subjected to the real-time PCR reaction using Taqman microRNA assay (Applied Biosystems). Each reaction was performed in triplicate. miR-103 was chosen as the endogenous control for signal normalization across different samples based on the recommendation of previous report . Normalized relative expression level of each miRNA was approximated by calculating 2-ΔCt (ΔCt = Ct_miRNA - Ct_miR-103, Ct: cycle threshold). Variation of mean Ct of miR-103 across four cord blood and four adult blood samples remained low (Avg_Ct = 19.75, Stdev = 1.09).
Results and Discussion
This report provides initial evidence that human let-7 miRNA, as a group, are up-regulated in association with fetal-to-adult hemoglobin switching. The erythroid focus of this study was chosen due to developmental similarities between fetal-to-adult transition in humans and related developmental changes in lower organisms. Also, miRNA expression patterns during late erythropoiesis were clinically associated with sickle cell anemia and malarial pathogenesis [20, 27]. While the results described here may be helpful for generating new hypotheses related to miRNA expression, more robust methods (including coordinated manipulation of multiple miRNA members) are needed to understand the functional significance of increased let-7 in adult erythroid cells. We speculate that let-7 or other differentially expressed miRNA are involved in the hemoglobin switching phenomenon. Alternatively, the increased let-7 expression in adult cells could affect other aspects of erythropoiesis since the predicted target genes are largely involved in cellular proliferation and apoptosis. Overall, these data strongly suggest that miRNA abundance patterns are developmentally regulated in circulating erythroid cells. As such, the data support further erythroid-focused investigation of these curious RNA molecules.
In addition to globin and other protein-encoding mRNA transcripts , miRNA species in circulating erythroid cells are differentially expressed in association with hemoglobin switching. Among the differentially-expressed miRNA, a majority of let-7 family members were significantly upregulated in adults. Differential expression of predicted let-7 target genes was also detected in the cells. Based upon the importance of let-7 for developmental transitions in lower organisms, it is proposed here that differential expression of miRNA including let-7 in erythroid cells should be explored for their potential to regulate changes in erythropoiesis or hemoglobin expression patterns in humans.
The Intramural Research Programs of the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and Clinical Center (Bethesda, MD) supported this research. We are additionally thankful for technical assistance from the NIDDK's microarray core facility.
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