1. miR-34a contributes to megakaryocytic differentiation of K562 cells independently of p53
by Francisco Navarro, David Gutman, Eti Meire, Mario Cáceres, Isidore Rigoutsos, Zvi Bentwich, and Judy Lieberman
Blood
Volume 114(10):
September 3, 2009
©2009 by American Society of Hematology

2. miRNA signature of TPA-treated K562 cells differentiated into megakaryocytes.
miRNA signature of TPA-treated K562 cells differentiated into megakaryocytes. (A) miRNAs up-regulated at least 6-fold 4 days after TPA treatment by microarray. Background counts were ≤ 500 and the assay saturated at 65 232 counts. Reproducibility of the miRNA microarray in 2 TPA-treated K562 samples is shown in supplemental Figure 1B. (Supplemental Table 1 shows data for all miRNAs analyzed.) (B-C) Change in miRNA expression after TPA or hemin treatment analyzed by Northern blot. U6 snRNA is a loading control. The miRNAs most induced by TPA were not induced by hemin (supplemental Figure 1C). MK differentiation by TPA and erythroid differentiation by hemin were verified (supplemental Figure 1A). miR-24 expression increased after either TPA or hemin treatment.
Francisco Navarro et al. Blood 2009;114:
©2009 by American Society of Hematology

3. miR-34a enhances megakaryocytic differentiation and inhibits proliferation in K562 cells.
miR-34a enhances megakaryocytic differentiation and inhibits proliferation in K562 cells. (A) Overexpression of miR-34a or miR-181a in K562 cells induces the MK integrin CD41. K562 cells were transfected with the indicated miRNA mimic and 24 hours later stimulated with a suboptimal amount of TPA (0.1 nM). Efficient uptake of dsRNA oligonucleotides into transfected K562 cells is shown in supplemental Figure 1D. CD41 expression was analyzed by flow cytometry 72 hours after TPA stimulation. As positive control, K562 cells were treated with 1 nM TPA. Levels of expression of the transfected miRNAs at the time of analysis are shown in supplemental Figure 2B. (B) miR-34a and miR-134 inhibit K562 cell proliferation, assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay. Cells were transfected with the indicated miRNA and then treated with a suboptimal amount of TPA as indicated. Values are mean ± SD.
Francisco Navarro et al. Blood 2009;114:
©2009 by American Society of Hematology

4. miR-34a and miR-134 inhibit cell-cycle progression at different phases of the cell cycle.
miR-34a and miR-134 inhibit cell-cycle progression at different phases of the cell cycle. K562 cells, transfected with the indicated miRNA mimic, were analyzed by propidium iodide staining 48 hours later. Replicate samples were either left untreated or treated with nocodazole (100 ng/mL) for 16 hours before propidium iodide staining. The percentage of cells in the G1, S, or G2 phase of the cell cycle is indicated. The bar graph at the bottom represents the mean ± SD of 4 independent experiments. #Significant differences (P < .05) relative to the control sample by Student 2-sided t test.
Francisco Navarro et al. Blood 2009;114:
©2009 by American Society of Hematology

5. miR-34a directly regulates MYB expression.
miR-34a directly regulates MYB expression. (A) miR-34a regulates luciferase activity of a reporter vector containing either the full-length 3′-UTR of MYB (left) or individual predicted MRE (right). Relative luciferase activity was assayed in 293T cells 48 hours after cotransfection with a luciferase reporter plasmid and the indicated miRNA mimic. AS-34a designates a reverse complementary sequence of miR-34a. (B) Overexpression of miR-34a in K562 cells down-regulates MYB protein and mRNA. K562 cells, transfected with the indicated miRNA mimic, were analyzed for MYB protein (left) and mRNA (right) by immunoblot or quantitative PCR, respectively, 48 hours later. Protein expression relative to α-tubulin was quantified by densitometry. mRNA normalized to GAPDH mRNA is shown relative to the ratio in K562 cells transfected with a miRNA mimic control. (C) MYB knockdown (right) up-regulates the MK-specific marker CD41 in cells treated with a suboptimal amount of TPA (0.1 nM). As positive control for CD41 induction, K562 cells were treated with 1 nM TPA.
Francisco Navarro et al. Blood 2009;114:
©2009 by American Society of Hematology

6. miR-34a regulates CDK4 and CDK6 in K562 cells.
miR-34a regulates CDK4 and CDK6 in K562 cells. (A) K562 cells, transfected with the indicated miRNA mimic, were analyzed for CDK4 and CDK6 protein and mRNA by immunoblot (left) or quantitative PCR (right), respectively, 48 hours later. Protein expression relative to α-tubulin was quantified by densitometry. mRNA normalized to GAPDH mRNA is shown relative to the ratio in K562 cells transfected with a miRNA mimic control. (B) Simultaneous knockdown (top left) of CDK4 and CDK6, but not either alone, inhibits proliferation and increases the G1 compartment in K562 cells. Cell proliferation was evaluated using the MTT cell proliferation assay (top middle). Cell-cycle analysis (representative flow cytometry histograms, bottom) was performed 72 hours after transfection in nocodazole-treated and untreated cells. The bar graph (top right) represents the mean ± SD of 4 independent experiments performed without nocodazole synchronization. Statistical analysis was performed using the Student 2-sided t test: #P < .05; **P < .01.
Francisco Navarro et al. Blood 2009;114:
©2009 by American Society of Hematology

7. Enforced miR-34a expression enhances MK differentiation of human CD34+ hematopoietic precursors.
Enforced miR-34a expression enhances MK differentiation of human CD34+hematopoietic precursors. (A) CD34+ hematopoietic cells, differentiated to MK by culture in medium containing TPO, SCF, and IL-3, express MK integrins. The proportion of CD41/CD61-positive cells is indicated. (B) Changes in miR-34a and its MYB, CDK4, and CDK6 target mRNAs were evaluated by quantitative PCR. miR-34a expression is normalized to U6 and mRNA is normalized to GAPDH. Gene expression is plotted as expression relative to day 0. ND indicates not detected. (C) CFU-MK assay of miRNA-overexpressing CD34+ HSCs. Primary human CD34+ cord blood cells were infected with lentiviruses encoding the indicated miRNAs, and the effect of miRNA overexpression on CFU-MK numbers was evaluated 12 days later. The bar graph represents the mean ± SD of quadruplicate samples. A representative experiment of 3 independent experiments is shown. #P < .05 relative to control lentivirus-infected cells. Exogenous miR-34a and miR-150 both enhance MK colony numbers.
Francisco Navarro et al. Blood 2009;114:
©2009 by American Society of Hematology

8. miR-34a up-regulation after TPA treatment of K562 cells is p53-independent.
miR-34a up-regulation after TPA treatment of K562 cells is p53-independent. (A) The p53-dependent miR-34a promoter does not activate luciferase expression after TPA treatment of K562 cells. Cells were transfected with a firefly luciferase reporter vector in which luciferase expression was driven by the DB miR-34a promoter (PM-34a) or the CD41 promoter (PM-CD41) or with the promoterless luciferase vector (pGL3-Basic). Samples were cotransfected with a Renilla luciferase reporter vector for normalization. Luciferase activity was measured 48 hours after TPA treatment. (B) Exogenous expression of p53 in K562 cells activates the DB miR-34a promoter. K562 cells were transfected with promoterless firefly luciferase reporter vector (pGL3) or a reporter containing the miR-34a promoter (PM-34a), a Renilla luciferase reporter vector, and increasing amounts (2, 4, and 8 μg) of pCMV-p53 vector. The total amount of transfected DNA was kept constant using an empty pCMV plasmid. Luciferase activity was measured 48 hours after transfection and was normalized as described. The fold increase in luciferase activity relative to the control sample transfected with the promoterless vector is shown. The bottom panel represents the level of p53 protein in the samples analyzed herein by immunoblot. The membrane was stripped and reprobed for α-tubulin as a loading control. (C) 5′- and 3′-end RACE PCR analysis of pri-miR-34a transcripts in TPA-treated K562 cells. Agarose gel images show the 5′- and 3′-RACE PCR products obtained in K562 cells treated with TPA for 4 days. (D) Two pri-miR-34a transcripts identified by sequencing the 5′-end RACE PCR products from TPA-treated K562 cells and their location in the genome. E indicates exon; the numbers below the exons indicate their length in base pairs. Also indicated is the length of the intronic regions. For comparison, the previously described pri-miR-34a transcript, DB286351, is also shown (top). (E) The genomic region adjacent to the alternate TSS serves as the miR-34a promoter in TPA-treated K562 cells. A DNA genomic fragment comprising 1.5 kb upstream to 0.5 kb downstream of the identified alternative TSS (PM-34a-K1/2) was cloned into pGL3-basic, and the effect of TPA treatment on luciferase activity was evaluated as described.
Francisco Navarro et al. Blood 2009;114:
©2009 by American Society of Hematology