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The Genomic Analysis of Erythrocyte microRNA Expression in Sickle Cell Diseases

Chen, Shao-Yin, Wang, Yulei, Telen, Marilyn J., Chi, Jen-Tsan·2008·PLoS ONE
DOI 10.1371/journal.pone.0002360PMC PMC2408759
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Figure 1 The purification of human mature erythrocytes. (A) The purification scheme used to obtain mature erythrocytes and reticulocytes from human blood. Assessment of the purified mature erythrocytes was then performed by examining the surface expression of indicated lineage markers on whole blood (before purification–blue line) and purified erythrocytes (after purification–red line) with FACS–CD16, CD45, CD71 and CD235a (B). (C) RT-PCR assay to evaluate the abundance of indicated transcripts (CD45, tRNA and beta-hemoglobin) in the RNA from purified erythrocytes (RBC), peripheral blood mononuclear cells (PBMC) and whole blood. (D) New methylene blue stain of the CD71- erythrocytes (left) and CD71+ reticulocytes (right) from two independent isolations.
Figure 1 The purification of human mature erythrocytes. (A) The purification scheme used to obtain mature erythrocytes and reticulocytes from human blood. Assessment of the purified mature erythrocytes was then performed by examining the surface expression of indicated lineage markers on whole blood (before purification–blue line) and purified erythrocytes (after purification–red line) with FACS–CD16, CD45, CD71 and CD235a (B). (C) RT-PCR assay to evaluate the abundance of indicated transcripts (CD45, tRNA and beta-hemoglobin) in the RNA from purified erythrocytes (RBC), peripheral blood mononuclear cells (PBMC) and whole blood. (D) New methylene blue stain of the CD71- erythrocytes (left) and CD71+ reticulocytes (right) from two independent isolations.CC BY · PubMed Central
Figure 2 Human mature erythrocytes contain abundant and diverse microRNAs. (A) The size distribution of RNAs of three independent erythrocyte samples (lanes 2–4 labeled RBC #1,2,3) and one PBMC sample (lane 5 labeled as PBMC) were assessed with Agilent Bioanalyzer with indicated size markers (lane 1 as indicated). (B) Left: the microRNA expression pattern obtained with microRNA microarrays of three mature erythrocyte samples was compared to that of two erythroleukemia K562 cell lines. Right: the expression of erythrocyte-specific microRNAs in the CD34+ erythroid progenitor cells at indicated stages of erythroid differentiation in a previously published study [14]. (C) The microRNA expression of whole blood was similar to erythrocytes and grouped together in one branch away from leukocytes with unsupervised hierarchical clustering. Selected erythrocyte-specific microRNAs were shown. (D) The expression of U6 and indicated microRNAs in K562 cells and erythrocytes was assessed by Northern blots with the migration position of U6 and mature microRNAs indicated. The position of U6 RNA and microRNAs (indicated bars on the right) were established with synthesized DNA oligonucleotides included in the Northern blots.
Figure 2 Human mature erythrocytes contain abundant and diverse microRNAs. (A) The size distribution of RNAs of three independent erythrocyte samples (lanes 2–4 labeled RBC #1,2,3) and one PBMC sample (lane 5 labeled as PBMC) were assessed with Agilent Bioanalyzer with indicated size markers (lane 1 as indicated). (B) Left: the microRNA expression pattern obtained with microRNA microarrays of three mature erythrocyte samples was compared to that of two erythroleukemia K562 cell lines. Right: the expression of erythrocyte-specific microRNAs in the CD34+ erythroid progenitor cells at indicated stages of erythroid differentiation in a previously published study [14]. (C) The microRNA expression of whole blood was similar to erythrocytes and grouped together in one branch away from leukocytes with unsupervised hierarchical clustering. Selected erythrocyte-specific microRNAs were shown. (D) The expression of U6 and indicated microRNAs in K562 cells and erythrocytes was assessed by Northern blots with the migration position of U6 and mature microRNAs indicated. The position of U6 RNA and microRNAs (indicated bars on the right) were established with synthesized DNA oligonucleotides included in the Northern blots.CC BY · PubMed Central
Figure 3 Erythrocyte microRNA show differentiation- and disease-specific expression. (A) The expression value of microRNA expression in the two reticulocytes (pink) and ten erythrocytes (red) assessed with TaqMan real-time assay. The expression levels of the 83 microRNAs identified by SAM as reticulocyte-specific were shown with selected microRNAs indicated. (B, C) The erythrocyte microRNA expression obtained from 7 HbAA (red) vs. 12 HbSS (brown) samples assessed with microRNA microarrays are distinct and grouped separately with unsupervised hierarchical clustering with several differentially expressed microRNAs indicated. (D) The average expression value of the 83 reticulocyte-specific microRNAs in the HbSS (brown) erythrocytes was significantly higher than that of HbAA (red) erythrocytes.
Figure 3 Erythrocyte microRNA show differentiation- and disease-specific expression. (A) The expression value of microRNA expression in the two reticulocytes (pink) and ten erythrocytes (red) assessed with TaqMan real-time assay. The expression levels of the 83 microRNAs identified by SAM as reticulocyte-specific were shown with selected microRNAs indicated. (B, C) The erythrocyte microRNA expression obtained from 7 HbAA (red) vs. 12 HbSS (brown) samples assessed with microRNA microarrays are distinct and grouped separately with unsupervised hierarchical clustering with several differentially expressed microRNAs indicated. (D) The average expression value of the 83 reticulocyte-specific microRNAs in the HbSS (brown) erythrocytes was significantly higher than that of HbAA (red) erythrocytes.CC BY · PubMed Central
Figure 4 CD71 downregulation during terminal differentiation is defective in HbSS reticulocytes and is a potential mRNA candidate for miR-320 regulation. (A) HbSS reticulocytes exhibited defective terminal differentiation. Four purified HbAA (red) and three purified HbSS (brown) reticulocyte samples were placed in differentiation culture media containing autologous plasma. The percentage of remaining CD71+ cells (normalized against the starting CD71+ percentage) after 24 and 48 hours was significantly higher for HbSS samples than HbAA samples (p = 0.0064). (B) The target sequence (red) of the CD71 3′UTR of the five indicated species was aligned with the seed sequence (blue) of miR-320. (C, D) The relative expression of miR-320 among the HbAA vs. HbSS erythrocytes is shown as heat map (C) or bar graph of centered mean expression values (D). (E) The relative expression of miR-320 in two reticulocyte (pink) and ten erythrocytes (red) samples was assessed with real-time PCR and shown.
Figure 4 CD71 downregulation during terminal differentiation is defective in HbSS reticulocytes and is a potential mRNA candidate for miR-320 regulation. (A) HbSS reticulocytes exhibited defective terminal differentiation. Four purified HbAA (red) and three purified HbSS (brown) reticulocyte samples were placed in differentiation culture media containing autologous plasma. The percentage of remaining CD71+ cells (normalized against the starting CD71+ percentage) after 24 and 48 hours was significantly higher for HbSS samples than HbAA samples (p = 0.0064). (B) The target sequence (red) of the CD71 3′UTR of the five indicated species was aligned with the seed sequence (blue) of miR-320. (C, D) The relative expression of miR-320 among the HbAA vs. HbSS erythrocytes is shown as heat map (C) or bar graph of centered mean expression values (D). (E) The relative expression of miR-320 in two reticulocyte (pink) and ten erythrocytes (red) samples was assessed with real-time PCR and shown.CC BY · PubMed Central
Figure 5 Poor expression of miR-320 in HbSS cells was associated with defective CD71 downregulation during terminal differentiation. (A) The histogram of the fluorescent intensity of reticulocytes transfected with unlabeled (black line) or fluorescently-labeled miR-320 (green line). (B) The miR-320 level in the transfected reticulocytes transfected with mock, synthesized miR-320 or LNA targeting miR-320 was determined with real-time PCR. (C, D) The percentage of remaining CD71+ cells normalized against the initial percentage after in vitro differentiation at indicated time points after transfection with either scrambled LNA or miR-320 knock down LNA. (E) Cell numbers of reticulocytes (left) and erythrocytes (right) 24 hours after transfection with treatments blocking indicated microRNAs were shown. (F). The reporter activities when empty vector or miR-451 and miR-320 expression constructs were co-transfected into K562 cells with respective indicator plasmids containing duplicated copies of sequences identical to miR-451 and mIR-320. (G) The over-expression of miR-320, but not miR-451 or empty vector, in K562 can suppress a reporter construct containing the 3′UTR of CD71. This suppression was abolished when three nucleotides in the miR-320 predicted target site were mutated as indicated in the 3′UTR of CD71. (H) Overexpression of miR-320 led to lower levels of CD71 in K562 cells 48 hours after transfection.
Figure 5 Poor expression of miR-320 in HbSS cells was associated with defective CD71 downregulation during terminal differentiation. (A) The histogram of the fluorescent intensity of reticulocytes transfected with unlabeled (black line) or fluorescently-labeled miR-320 (green line). (B) The miR-320 level in the transfected reticulocytes transfected with mock, synthesized miR-320 or LNA targeting miR-320 was determined with real-time PCR. (C, D) The percentage of remaining CD71+ cells normalized against the initial percentage after in vitro differentiation at indicated time points after transfection with either scrambled LNA or miR-320 knock down LNA. (E) Cell numbers of reticulocytes (left) and erythrocytes (right) 24 hours after transfection with treatments blocking indicated microRNAs were shown. (F). The reporter activities when empty vector or miR-451 and miR-320 expression constructs were co-transfected into K562 cells with respective indicator plasmids containing duplicated copies of sequences identical to miR-451 and mIR-320. (G) The over-expression of miR-320, but not miR-451 or empty vector, in K562 can suppress a reporter construct containing the 3′UTR of CD71. This suppression was abolished when three nucleotides in the miR-320 predicted target site were mutated as indicated in the 3′UTR of CD71. (H) Overexpression of miR-320 led to lower levels of CD71 in K562 cells 48 hours after transfection.CC BY · PubMed Central

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Metadata

Authors
Chen, S.-Y., Wang, Y., Telen, M. J., & Chi, J.-T.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2008-06-04
Volume
3
Issue
6
Pages
e2360
ISSN
1932-6203
DOI
10.1371/journal.pone.0002360
PMC
PMC2408759
Cited by
163
References
2
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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