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Blood, Vol. 114, Issue 10, 2181-2192, September 3, 2009
miR-34a contributes to megakaryocytic differentiation of K562 cells independently of p53
Blood Navarro et al.
114: 2181
Supplemental materials for: Navarro et al
RNA extraction and Northern blot
Total RNA was isolated using Trizol (Invitrogen). RNA samples (40 µg) were resolved on 15% TBE-Urea acrylamide gels (Invitrogen), transferred onto Nytran-SPC Nylon Membranes (Whatman) and UV-crosslinked. Membranes were prehybridized at 42°C in 7% SDS/0.2 M sodium phosphate buffer and hybridized at 42°C with α32P-dATP 3′–end-labeled miRNA DNA oligonucleotide probes containing a reverse and complementary miRNA sequence (Integrated DNA Technologies, IDT). After washing twice at room temperature with 2× SSC/0.1% SDS the membranes were analyzed by PhosphorImager (Storm 860, GE Healthcare). Membranes were also probed for U6 snRNA as loading control. Flow cytometry and proliferation assays
Indirect immunostaining of CD41 and CD61 was performed using mouse mAb (BD Biosciences) and phycoerythrin-conjugated sheep anti-mouse Ig (Caltag) secondary antibody. Samples were analyzed by FACSCalibur and CellQuest software (BD Biosciences). For cell cycle analysis, cells were washed once in PBS (Invitrogen), fixed in 70% ethanol at 4°C, washed again in PBS and resuspended in Propidium Iodide (PI) staining solution (0.1% (v/v) Triton X-100 in PBS, containing 200 µg/ml DNase-free RNase A and 20 µg/ml PI (Molecular Probes). Samples were incubated at 37°C for 15 min before flow cytometry analysis using FlowJo (Tree Star, Inc). Proliferation assays were performed in quadruplicate using the MTT Cell Proliferation Assay (ATCC). Plasmid DNA and lentiviruses
The full length MYB 3′UTR was cloned into the XbaI/SalI restriction sites of the pGL3-control luciferase reporter vector (Promega), replacing the SV40 enhancer and SV40 poly(A) signal. For testing predicted MREs, sense and antisense oligomers were synthesized, annealed, and cloned into psiCHECK-2 (Promega) at XhoI and NotI restriction sites, directly downstream of Renilla luciferase. The plasmid pCMV6-TP53, encoding the full length p53 cDNA, was obtained from Origene. The sequence of the promoter region of miR-34a based on Raver-Shapira et al,1 was PCR-amplified from K562 cell genomic DNA and cloned into the NheI and XhoI restriction sites of pGL3-basic (Promega). The sequence corresponding to the previously described CD41 promoter2 was also cloned into pGL3-basic using the same restriction sites. Short hairpin RNA (shRNA) constructs were generated using the pSIREN-RetroQ vector (Clontech) following the manufacturer’s instructions. siRNAs were designed using “siRNA Target Finder” (Ambion). Sequences were: c-MYB: sense strand, 5′-GCGAATAAAGGAATTAGAA-3′, antisense strand, 5′-TTCTAATTCCTTTATTCGC-3′; CDK4: sense strand, 5′-GGTAACCCTGGTGTTTGAG-3′, antisense strand, 5′-CTCAAACACCAGGGTTACC-3′; CDK6: sense strand, 5′-CATGTCGATCAAGACTTGA-3′, antisense strand, 5′-TCAAGTCTTGATCGACATG-3′. Lentiviruses were generated using the pLL3.7 vector (generously provided by L. Van Parijs). The pLL3.7 cassette containing the U6 promoter and a control short hairpin was replaced by a cassette containing the U6 promoter and an approx 300 bp genomic fragment for expression of the mature miRNA from pSIREN-RetroQ-miRNA expression vectors (kind gift of B. Ramratnam, Brown University). Lentivirus stocks were generated by co-transfecting 293T cells with the lentiviral construct, pLL3.7-miRNA, plus pΔR8.9 and pVSV-G. Virus supernatants were collected 48 hours post-transfection, filtered, and concentrated by ultracentrifugation. Immunoblot
Total cell extracts were prepared in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml leupeptin. Protein concentration was measured using the BCA Protein Assay Reagent (Pierce) and 4 µg of total protein were resolved on 10% SDS-PAGE, transferred to PVDF membranes (Immobilon-P, Millipore), and probed with mouse mAb against MYB (Upstate), CDK4, or CDK6 (Cell Signaling Technology) followed by horseradish peroxidase-(HRP) conjugated sheep anti-mouse Ig (Amersham). The membranes were developed using SuperSignal West Femto (Pierce). For loading control, the membranes were stripped and reprobed using α-tubulin mouse mAb (Sigma-Aldrich). Immunoblots were quantified by densitometry using VersaDoc MP 4000 (Bio-Rad). Luciferase assay
The luciferase activity of reporter plasmid-transfected samples was measured 48 hr post-transfection using the Dual Luciferase Assay System (Promega) and a Top count NXT microplate reader (Perkin Elmer) as per manufacturer’s instructions. Data were normalized relative to Firefly or Renilla luciferase. Statistical analysis
Data were analyzed by 2-sided student’s t-test. P-values <0.05 were considered significant. REFERENCES 1. Raver-Shapira N, Marciano E, Meiri E, et al. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell. 2007;26:731–743.
2. Sevinsky JR, Whalen AM, Ahn NG. Extracellular signal-regulated kinase induces the megakaryocyte GPIIb/CD41 gene through MafB/Kreisler. Mol Cell Biol. 2004;24:4534–4545.
Files in this Data Supplement:
- Table S1. miRNA expression in TPA-treated vs. untreated K562 cells by miRNA microarray (PDF, 153 KB)
- Table S2. Changes in gene expression in TPA-treated vs. untreated K562 cells by gene microarray (PDF, 125 KB) -
RNA was harvested 2 days after TPA-treatment and compared with RNA from untreated cells. Genes whose expressed varied by at least 2-fold are listed.
- Table S3. Gene expression alterations in miR-34a vs. control transfected K562 cells by gene microarray (PDF, 186 KB) -
RNA was harvested 24 hr after transfection with miR-34a or a control miRNA mimic. Genes whose expression varied by at least 1.9-fold are listed.
- Figure S1. Differentiation of K562 cells (JPG, 256 KB)
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(A) TPA drives MK differentiation and hemin drives erythrocyte differentiation of K562 cells. Differentiation status of TPA- or hemin-treated K562 cells by flow cytometry analysis of CD41 expression (left) or benzidine staining (right), respectively. (B) Correlation of miRNA expression assessed by miRNA microarray 2 and 4 days following TPA treatment. Linear regression plot of D2/D0 ratio vs. D4/D0 ratio of relative miRNA expression in K562 cells harvested after 2 or 4 days of TPA treatment. (C) miR-134 is not modulated during hemin-induced erythrocyte differentiation of K562 cells. miR-134 levels in hemin or TPA treated K562 samples were analyzed by qRT-PCR and compared to the levels in untreated cells. The data, normalized to U6, are the mean ± S.D. of quadruplicate samples. A representative experiment of three performed is shown. (D) Efficient uptake of dsRNA oligonucleotides by K562 cells. To analyze transfection efficiency, cells were transfected by nucleofection with 100 nM of a FITC-conjugated CCR5 siRNA or left untreated and analyzed by flow cytometry 24 hours post-transfection. (E) miR-150 is not detected by Northern blot in untreated or TPA-treated K562 cells. Northern blot analysis of miR-150 expression in untreated or TPA-treated K562 cells after 2 or 4 days. To control for hybridization, RNA from 293T cells transfected with pLL3.7-miR-150 was analyzed. The samples were also blotted for miR-34a, miR-25, and U6 as additional controls.
- Figure S2. Effect of miRNA over-expression on MK differentiation of K562 cells (JPG, 247 KB)
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(A) K562 cells were transfected with the indicated miRNA mimics and 24 h later were stimulated with a sub-optimal amount of TPA (0.1 nM). CD41 expression was analyzed by flow cytometry 72 h post-TPA stimulation. As a positive control for CD41 expression, K562 cells were treated with 5 nM TPA. (B) Northern blot analysis of K562 cells that were untransfected or transfected with miR-34a, miR-181a or control C. elegans miRNA (cel-miR-67) harvested 24 hours post-transfection and 72 hours post-treatment with a suboptimal amount of TPA. Samples were processed as described in Fig. 2.
- Figure S3. Mutation or deletion of MYB miR-34a MRE2 partially restores the luciferase activity of the pGL3-MYB-3′UTR luciferase reporter vector (JPG, 222 KB)
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(A) Complementarity of the rna22-predicted MREs and miR-34a. G-U wobble base pairing is indicated by red dots. Mutations introduced into the seed of the MRE2 binding site are indicated in red. The numbers in brackets indicate their location in the 3′UTR of MYB. (B) Mutation or deletion of MRE2 partially restores luciferase activity of the pGL3-MYB-3′UTR reporter vector. The data are the mean ± S.D. of ten independent experiments. Statistically significant differences between wt and MRE2-Mut or MRE2-Del are indicated by p values obtained using the Student’s t-test.
- Figure S4. miR-221 and miR-134 directly regulate MYB expression in K562 cells (JPG, 246 KB)
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(A) Immunoblot analysis of MYB protein levels in miRNA-overexpressing K562 cells (top). Control cells were transfected with cel-miR-67 (Dharmacon) which has no homology with human genes. TargetScan does not predict miR-25 and miR-375 as regulators of MYB. The bar graph at the bottom shows the relative ratio of the normalized signal (normalized to α-tubulin) determined by densitometry and represented as the fraction of the control corresponding to the mean ± S.D. of three independent experiments. #, p<0.05; **, p<0.01. (B) Effect of miRNA over-expression on the activity of a luciferase reporter vector containing the full length MYB 3′UTR. Relative luciferase activity was assayed in K562 cells 48 h after co-transfection with a luciferase reporter plasmid and the indicated miRNA mimic. The bar graph represents the mean ± S.D. of three independent experiments. #, p<0.05; **, p<0.01 relative to control samples transfected with cel-miR-67.
- Figure S5. Analysis of CDK6 rna22-predicted miR-34a MREs by luciferase assays (JPG, 347 KB)
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(A) Table showing the CDK6 MREs predicted by rna22 for miR-34a. Indicated is their location in the 3′UTR and the values of the folding energy of the heteroduplex (Kcal/mol). G-U wobble base pairing is indicated by red dots. MRE3 is also predicted by TargetScan. (B) Analysis of effect of miR-34a over-expression on the luciferase activity of reporter vectors containing the individual rna22-predicted MREs. The analysis was performed as described previously. The diagram represents the percentage of luciferase activity relative to the control (psiCHECK2 vector with no MRE).
- Figure S6. Kinetic analysis of the expression of miR-34a and its targets in TPA-treated K562 cells (JPG, 229 KB)
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(A) Kinetics of down-regulation of MYB, CDK4, and CDK6 proteins after TPA-treatment. K562 cells were treated with 10 nM TPA and protein expression of MYB, CDK4, and CDK6 were determined by immunoblot analysis at the indicated times post-treatment. The membranes were stripped and reprobed with an Ab against α-tubulin as loading control. (B) Kinetics of expression of MYB mRNA and miR-34a after TPA treatment of K562 cells by Northern blot. As loading control the membranes were stripped and reprobed for β-actin and U6, respectively. (C) Quantification of the time course expression of miR-34a, MYB, CDK4, and CDK6. Protein expression was quantitated by densitometry and normalized to α-tubulin. The data are represented as the percentage relative to the untreated sample. miR-34a expression, normalized to U6, was determined by PhosphorImager analysis, and the data were plotted as the percentage of the signal obtained at 96 hours.
- Figure S7. RACE PCR analysis of pri-miR-34a transcript in TPA-treated K562 cells (JPG, 240 KB)
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(A) Diagram depicting the location, on the annotated EST DB286351, of the primers used in the 5′/3′ RACE PCR analysis. (B) Sequence analysis of the products obtained from the 5′ and 3′ end RACE PCR amplication of the pri-miR-34a transcript in TPA-treated K562 cells. All the clones sequenced from the 5′ end RACE PCR analysis (14 in total) started at the same position indicated in red and with an asterisk. The results obtained from the 3′ end RACE indicated more heterogeneity. The nucleotide at which the transcripts terminated is indicated in red and with an asterisk. The number in parenthesis indicates the number of clones that ended at the indicated position.
- Figure S8. Sequence of the pri-miR-34a transcripts identified in TPA-treated K562 cells (JPG, 530 KB)
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Indicated is the sequence of the pri-miR-34a transcripts identified in TPA-treated K562 cells by RACE PCR analysis. The first nucleotide in red at the 5′ end indicates the TSS. Exon boundaries are indicated by bases in bold and arrows. The nucleotide in red at the 3′ end indicates the end of transcription of the annotated EST DB286351.
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