The Genomic Analysis of Erythrocyte microRNA Expression in Sickle Cell Diseases

Abstract

BackgroundSince mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS).Methods and FindingsUsing a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation.ConclusionsIn summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases.

MicroRNA signature of inflamed lymphatic endothelium and role of miR-9 in lymphangiogenesis and inflammation

“…The cycle threshold (2 Ϫ⌬⌬Ct ) method was utilized to calculate the fold change. As normalization of the miRNA data is critical in elimination of the bulk of false positives, the most stably expressed genes across the arrays, including three of the manufacturer-provided housekeeping genes (U6, 4.5S, and Y1) and miR152, were used to normalize the expression data; expression of these genes was unchanged across the arrays and time points as determined by NormFinder (8).…”

MicroRNA 223-dependent expression of LMO2 regulates normal erythropoiesis

“…Several miRNAs participate in the regulation of erythropoiesis: miR-221/222 inhibit normal erythropoiesis and erythroleukemic cell growth down-regulating kit receptor expression, 28 miR-24 hampers erythropoiesis by targeting ALK4 and perturbing activin signaling, 39 miR-320 finely tunes the translational activities of CD71 in reticulocytes, 20 c-Myb and miR-15a form an autoregulatory feedback loop which govern the transition from BFU-E to CFU-E stage. 40 Our results suggest that also miR-223 belongs to a functional set of miRNAs implicated in fine-tuning the expression of key genes to a physiologically relevant level during erythropoiesis.…”

“…[13][14][15] The commitment of pluripotent precursors to the erythroid lineage and the differentiation of committed erythroid progenitors into erythrocytes are associated with the induction or repression of several miRNAs. [16][17][18][19][20] However, the majority of their target genes are predicted computationally and only a few targets have been validated so far, preventing delineation of miRNAbased control circuitries.…”

MicroRNA levels quantified in whole blood varies from PBMCs

“…Due to the unique miRNA expression pattern in each cell-type the relative proportions of cells in blood may have an effect on the overall miRNA expression profile and the expression of their protein targets ( Min et al , 2010). Some studies have shown that mature miRNA expression signature in erythrocytes is similar to that in whole blood while different when compared to PBMCs ( Chen et al , 2008). MiR-451-5p is a marker of erythrocytes ( Rasmussen et al , 2010) and miR-23a-3p is unaffected by haemolysis ( Blondal et al , 2013).…”