Acute myeloid leukemia cells secrete microRNA-4532-containing exosomes to mediate normal hematopoiesis in hematopoietic stem cells by activating the LDOC1-dependent STAT3 signaling pathway

Abstract

Background: MicroRNA (miR)-containing exosomes released by acute myeloid leukemia (AML) cells can be delivered into hematopoietic progenitor cells to suppress normal hematopoiesis. Herein, our study was performed to evaluate the effect of exosomal miR-4532 secreted by AML cells on hematopoiesis of hematopoietic stem cells.

Methods: Firstly, differentially expressed miRs related to AML were identified using microarray analysis. Subsequently, AML cell lines were collected, and CD34⁺ HSCs were isolated from healthy pregnant women. Then, miR-4532 expression was measured in AML cells and AML cell-derived exosomes and CD34⁺ HSCs, together with evaluation of the targeting relationship between miR-4532 and LDOC1. Then, AML cells were treated with miR-4532 inhibitor, and exosomes were separated from AML cells and co-cultured with CD34⁺ HSCs. Gain- and loss-function approaches were employed in CD34⁺ HSCs. Colony-forming units (CFU) and expression of dickkopf-1 (DKK1), a hematopoietic inhibiting factor associated with pathogenesis of AML, were determined in CD34⁺ HSCs, as well as the extents of JAK2 and STAT3 phosphorylation and LDOC1 expression.

Results: miR-4532 was found to be upregulated in AML cells and AML cell-derived exosomes, while being downregulated in CD34⁺ HSCs. In addition, exosomes released by AML cells targeted CD34⁺ HSCs to decrease the expression of CFU and increase the expression of DKK1. miR-4532 was delivered into CD34⁺ HSCs to target LDOC1 via AML cell-released exosomes. AML cell-derived exosomes containing miR-4532 inhibitor increased CFU but reduced DKK1 in CD34⁺ HSCs. Inhibition of miR-4532 or JAK2, or ectopic expression of LDOC1 upregulated CFU and downregulated DKK1 expression as well as the extents of JAK2 and STAT3 phosphorylation in CD34⁺ HSCs.

Conclusion: In conclusion, AML cell-derived exosomes carrying miR-4532 repress normal HSC hematopoiesis via activation of the LDOC1-dependent STAT3 signaling pathway.

Keywords: Acute myeloid leukemia; Exosomes; Hematopoiesis; Hematopoietic stem cells; LDOC1; STAT3 signaling pathway; miR-4532.

Fig. 1

miR-4532 expression is upregulated in AML cell-secreted exosomes. a Screening of AML-related miRs in the GSE85769 microarray data. The X axis represents the sample number, and the Y axis represents the gene name. The histogram on the upper right is color gradation, with each rectangle representing a corresponding sample expression value. b miR-4532 expression in AML cell lines (HL-60, Molm-14, ML-2, and OCI-AML3) and CD34⁺ HSCs, as measured by RT-qPCR. *p < 0.05 vs. CD34⁺ HSCs. c Schematic diagram of exosome separation procedure. To exclude the effect of exosomes on bovine, the medium or FBS was centrifuged at 100,000×g for 10 h to remove the bovine exosomes. After that, the centrifugal medium was filtered through a 0.2-μm filter and collected for cell culture. AML cell line was cultured with the centrifugal medium. After 48 h, the supernatant was obtained. The process of exosome separation is shown in c. Finally, the purified exosomes were rinsed twice with PBS. d Morphology of exosomes observed under TEM. e The size and concentration of exosomes evaluated by nanoparticle tracking analysis. f Identification of marker exosomes (TSG101, CD63, and histone) by Western blot analysis. g miR-4532 expression in AML cell lines (HL-60, Molm-14, ML-2, and OCI-AML3) and exosomes secreted from AML cell lines (HL-60, Molm-14, ML-2, and OCI-AML3), as measured by RT-qPCR. *p < 0.05 vs. HL-60 cells, Molm-14 cells, ML-2 cells, or OCI-AML3 cells. Measurement data were described as mean ± standard deviation. Comparisons between two groups were analyzed by unpaired t test, and comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The cell experiment was repeated three times to obtain the mean value. miR-4532, microRNA-4532; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TEM, transmission electron microscope; PBS, phosphate buffer saline; FBS, fetal bovine serum

Fig. 2

AML cell-secreted exosomes target HSCs to mediate hematopoiesis. a, b Uptake of CFSE-labeled exosomes by CD34⁺ HSCs, as detected under the fluorescence microscope. (×400) c Colony-forming capacity of CD34⁺ HSCs 48 h after culture with different dose of Molm-14 cell-derived exosomes. d Relative expression of hematopoietic suppressor gene DKK1 in CD34⁺ HSCs after culture with different dose of Molm-14 cell-derived exosomes measured by RT-qPCR. *p < 0.05 vs. the treatment of PBS. Measurement data were described as mean ± standard deviation. Comparisons between two groups were analyzed by unpaired t test. The cell experiment was repeated three times to obtain the mean value. miR-4532, microRNA-4532; LDOC1, leucine-zipper downregulated in cancer 1; DKK1, dickkopf-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-qPCR, reverse transcription quantitative polymerase chain reaction

Fig. 3

AML cells transfer miR-4532 by exosomes to target LDOC1. a Venn diagram of the predicted target genes of miR-4532. b The targeting relationship between miR-4532 and LDOC1 in HEK-293 T cells and CD34⁺ HSCs verified by dual-luciferase reporter gene assay (*p < 0.05 vs. the co-treatment of mimic-NC and LDOC1 3′-UTR WT). c LDOC1 expression in AML-related microarray data GSE9476. d LDOC1 expression in AML retrieved from the TCGA database (the X axis indicates the sample type, and the Y axis indicates the expression value; the red box indicates the tumor sample, and the gray box indicates the normal sample; *q < 0.01). e LDOC1 expression in AML cells and CD34⁺ HSCs measured by RT-qPCR (*p < 0.05 vs. CD34⁺ HSCs). f miR-4532 expression in Molm-14 cells after transfection with miR-4532 inhibitor measured by RT-qPCR (*p < 0.05 vs. Molm-14 cells cultured with miR-4532 inhibitor). g miR-4532 expression in the exosomes from Molm-14 cells measured by RT-qPCR (*p < 0.05 vs. exosomes from inhibitor-NC-treated Molm-14 cells). h miR-4532 and LDOC1 expression in CD34⁺ HSCs after co-culture with the exosomes from transfected Molm-14 cells measured by RT-qPCR (*p < 0.05 vs. CD34⁺ HSCs treated with PBS, ^#p < 0.05 vs. CD34⁺ HSCs co-cultured with exosomes from inhibitor-NC-treated Molm-14 cells). Measurement data were described as mean ± standard deviation. Comparisons between two groups were analyzed by unpaired t test, and comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The cell experiment was repeated three times to obtain the mean value. miR-4532, microRNA-4532; LDOC1, leucine-zipper downregulated in cancer 1; RT-qPCR, reverse transcription quantitative polymerase chain reaction

Fig. 4

AML cell-derived exosomes overexpressing miR-4532 suppress normal hematopoiesis by targeting LDOC1. a The colony-forming capacity of CD34⁺ HSCs 48 h after culture with transfected Molm-14 cell-derived exosomes. b The relative expression of hematopoietic suppressor gene DKK1 in CD34⁺ HSCs after culture with transfected Molm-14 cell-derived exosomes measured by RT-qPCR. *p < 0.05 vs. CD34⁺ HSCs co-cultured with exosomes from inhibitor-NC-treated Molm-14 cells. c Colony-forming capacity of CD34⁺ HSCs after alteration of miR-4532 and LDOC1. d The relative expression of hematopoietic suppressor gene DKK1 in CD34⁺ HSCs after alteration of miR-4532 and LDOC1 measured by RT-qPCR. *p < 0.05 vs. CD34⁺ HSCs treated with mimic-NC, ^#p < 0.05 vs. CD34⁺ HSCs cultured with miR-4532 mimic, ^&p < 0.05 vs. CD34⁺ HSCs cultured with si-NC. Measurement data were described as mean ± standard deviation. Comparisons between two groups were analyzed by unpaired t test, and comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The cell experiment was repeated three times to obtain the mean value. miR-4532, microRNA-4532; LDOC1, leucine-zipper downregulated in cancer 1; RT-qPCR, reverse transcription quantitative polymerase chain reaction

Fig. 5

miR-4532 upregulation represses hematopoiesis of HSCs by activating LDOC1-dependent STAT3 signaling pathway. a The extents of JAK2 and STAT3 phosphorylation in CD34⁺ HSCs treated with different dose of exosomes, as determined by Western blot analysis (*p < 0.05 vs. CD34⁺ HSCs treated with PBS). b The extents of JAK2 and STAT3 phosphorylation in CD34⁺ HSC culture with transfected Molm-14 cell-derived exosomes, as determined by Western blot analysis (*p < 0.05 vs. CD34⁺ HSCs co-cultured with exosomes from inhibitor-NC-treated Molm-14 cells). c The extents of JAK2 and STAT3 phosphorylation in CD34⁺ HSCs after alteration of miR-4532 and LDOC1 determined by Western blot analysis (*p < 0.05 vs. CD34⁺ HSCs treated with mimic-NC, ^#p < 0.05 vs. CD34⁺ HSCs cultured with miR-4532 mimic, ^&p < 0.05 vs. CD34⁺ HSCs cultured with si-NC). d The extents of JAK2 and STAT3 phosphorylation in CD34⁺ HSCs after inhibition of JAK2 determined by Western blot analysis. e Colony-forming capacity of CD34⁺ HSCs after inhibition of JAK2. f The relative expression of hematopoietic suppressor gene DKK1 in CD34⁺ HSCs after inhibition of JAK2 measured by RT-qPCR. *p < 0.05 vs. CD34⁺ HSCs treated with DMSO. Measurement data were described as mean ± standard deviation. Comparison between two groups was analyzed by unpaired t test, and comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The cell experiment was repeated three times to obtain the mean value. miR-4532, microRNA-4532; LDOC1, leucine-zipper downregulated in cancer 1; RT-qPCR, reverse transcription quantitative polymerase chain reaction; STAT3, signal transducer and activator of transcription 3

Fig. 6

The mechanism of AML cell-secreted exosomal miR-4532 in hematopoiesis of HSCs. AML cell-secreted exosomes transfer miR-4532 into HSCs, and miR-4532 targets and downregulates LDOC1, thus activating the STAT3 signaling pathway. The activation of the STAT3 signaling pathway increased the hematopoietic suppressor gene DKK1 expression, thus suppressing hematopoiesis of HSCs. miR-4532, microRNA-4532; LDOC1, leucine-zipper downregulated in cancer 1; STAT3, signal transducer and activator of transcription 3