Erythroid cells (reticulocytes and mature erythrocytes) were isolated and purified from blood. The strategies used to isolate the erythroid cells in high purity (>99% erythroid cells in the absence of leukocytes and platelets) were previously described [12
]. Total RNA was isolated within 48 hours of collection from fetal (umbilical cord, n = 4) and adult (n = 4) blood sources. Among the 474 human miRNAs spotted on the arrays, 206 were detected in the samples. As defined by p < 0.01 and mean fold change > 2, 41 miRNA species were identified as being differentially expressed in the fetal and adult cells. According to these criteria, only 4 of 41 miRNAs demonstrated significantly down-regulated abundance in the adult cells, and none were down-regulated to levels below a negative three-fold change. The remaining 37 of the 206 human miRNAs were upregulated in abundance in the adult samples. Among the up-regulated subgroup, hsa-miR-96 demonstrated a distinct pattern with a 34.4 fold increase in abundance. Also noteworthy were hsa-miR-411 with a 7.5 fold increase, hsa-miR-182 with a 5.1 fold increase, and hsa-let-7
miRNAs with 4.3 to 5.1 fold increases (Figure ). The unbalanced pattern of up-regulation compared to down-regulation in the adult samples was opposite the pattern of mRNA previously reported among similar erythroid populations [12
]. In that study, the fetal erythroid cells were identified as having increased abundance in 103 of 107 differentially regulated mRNAs. The cause of increased abundance of miRNA versus decreased mRNA abundance in the adult cells is unknown, but the pattern is consistent with the general role of miRNA for mRNA degradation.
Figure 1 MicroRNA expression profiles of reticulocytes from cord blood and adult blood samples. Total RNA was isolated from enucleated reticulocyte-enriched pools from four umbilical cord blood samples (CB) and four adult peripheral blood samples (AB). Raw intensities (more ...)
In order to validate the array-based patterns of human erythroid miRNA, qPCR assays were performed. Relative abundance of miRNA in each sample was calculated by delta Ct method using miR-103 as a reference [14
]. Equivalent and high-level expression of miR-103 was detected in cord and adult blood samples (data not shown). The pattern of increased let-7
miRNA abundance demonstrated on the arrays was confirmed by qPCR (Figure ). Among the let-7
miRNA detected on the arrays with significantly increased abundance, let-7d
miRNA demonstrated the greatest increases with more than 10 fold increases with qPCR (p < 0.01). Differential expression of let-7f
was not identified by qPCR, and let-7b failed to amplify. In addition to the let-7
miRNA group, qPCR was also used to confirm the expression patterns of other miRNA in these cells. Increased abundance of three other up-regulated miRNA (miR-96, miR-29c, and miR-429) was confirmed (Figure ). miR-96 was the most differentially expressed on the arrays, and the qPCR data confirmed greater than a 10-fold increase in adult cells. Up-regulated expression of miR-96 was recently demonstrated in chronic myelogenous leukemia and breast cancer cells [15
], and miR-96 may function by regulating expression of the transcription factor FOXO1 [16
]. The expression patterns of three other miRNA (miR-451, miR-144, and miR-142) predicted to be expressed in erythroid cells were also examined (Figure ). miR-142 is specifically expressed in hematopoietic tissues [17
]. The miR-144 and miR-451 genes are known erythroid miRNA that are regulated by the GATA-1 transcription factor [18
]. All three miRNA species were detected. Adult blood expression of miR-451 was increased, but that increase was not statistically significant.
Figure 2 Validation of miRNA array data using quantitative real-time polymerase chain reaction (qPCR) assay. A. Relative expression patterns for the let-7 miRNA that were quantitated by qPCR. Relative expression levels (y-axis) in umbilical cord blood were defined (more ...)
While the expression of let-7 genes in human erythroid cells was reported previously [20
], this is the first study to demonstrate a developmental increase in the abundance of these gene products. Since let-7
miRNA is involved in ontogeny-related gene expression and regulation in lower organisms [8
], our study was extended to identify potential mRNA targets of let-7
that are expressed in fetal versus adult human erythroid cells. For this purpose, the miRNA expression patterns were combined with mRNA transcriptome analyses. First, miRBase predictions (Version 5) of let-7 major strands were catalogued according to a prediction p-value of less than 0.001. In total, 532 human genes were identified as potential targets of the differentially expressed let-7 miRNA shown in Figure . Next, mRNA profiling analyses were performed on the circulating erythroid cells to determine which of the target genes demonstrated down-regulated abundance in the adult cells. Among 532 target genes, the mRNA levels of 10 predicted gene targets were down-regulated in adult blood compared to umbilical cord blood (Figure ). Collectively, the group includes several genes involved in cellular proliferation (MED28, SMOX) [21
], and apoptosis (DAD1, EIF4G2) [23
]. Also, EIF3S1 [25
] functions in the 40S ribosomal initiation complex formation, so down-regulation of this non-core subunit of EIF3 may affect erythroblast differentiation or the translational efficiency of globin chain mRNAs [26
]. Unlike the model organisms like C. elegans, there was little evidence suggesting let-7 significantly regulates Ras mRNA in these human cells.
Figure 3 Reticulocyte mRNA expression levels of 10 genes that are predicted targets of let-7 miRNA. Average intensities of each probe set for let-7 target genes in umbilical cord blood versus adult blood were calculated from mRNA expression profiling data using (more ...)
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
]. 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.