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miR-17 promotes expansion and adhesion of human cord blood CD34(+) cells in vitro.

Yang Y, Wang S, Miao Z, Ma W, Zhang Y, Su L, Hu M, Zou J, Yin Y, Luo J - Stem Cell Res Ther (2015)

Bottom Line: However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells.The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical & Research Technology, School of Medicine, University of Maryland, Baltimore, MD, 21201, USA. yangyx@bjmu.edu.cn.

ABSTRACT

Introduction: We have recently found that miR-17 is necessary in the cell-extrinsic control of cord blood (CB) CD34(+) cell function. Here, we demonstrated that the proper level of miR-17 is also necessary in the cell-intrinsic control of the hematopoietic properties of CB CD34(+) cells.

Methods: The miR-17 overexpression and knockdown models were created using primary CB CD34(+) cells transfected by the indicated vectors. Long-term culture, colony forming, adhesion and trans-well migration assays were carried out to investigate the function of miR-17 on CB CD34(+) cells in vitro. NOD prkdc (scid) Il2rg () mice were used in a SCID repopulating cell assay to investigate the function of miR-17 on CB CD34(+) cells in vivo. A two-tailed Student's t-test was used for statistical comparisons.

Results: In vitro assays revealed that ectopic expression of miR-17 promoted long-term expansion, especially in the colony-forming of CB CD34(+) cells and CD34(+)CD38(-) cells. Conversely, downregulation of miR-17 inhibited the expansion of CB CD34(+) cells. However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells. The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.

Conclusion: We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

No MeSH data available.


Related in: MedlinePlus

The expression of miR-17 in CB hematopoietic CD34+CD38−/CD38+ cells. a The expression level of miR-17 in CB CD34+CD38−/CD38+ cells was evaluated by real-time PCR. Each reaction was performed in triplicate. Student’s t-test was used for the statistical analysis between the two indicated populations. b Real-time PCR was performed to evaluate the expression level of miR-17 in CB CD34+ cells after transfection with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD or 17/KD1), or control (CTRL). The data are presented as the ratio of miR-17 levels (relative to U6) in 17/OE, 17/KD or 17/KD1 to that in CTRL. Each reaction was performed in triplicate. c The expression level of miR-17 in 17/OE CD34+ cells or 17/KD CD34+ cells 5 days after sorting was evaluated by real-time PCR. d The expression level of miR-17 in 17/OE CD34+CD38−/CD38+ cells or 17/KD CD34+CD38−/CD38+ cells 20 days after culture was evaluated by real-time PCR
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Fig1: The expression of miR-17 in CB hematopoietic CD34+CD38−/CD38+ cells. a The expression level of miR-17 in CB CD34+CD38−/CD38+ cells was evaluated by real-time PCR. Each reaction was performed in triplicate. Student’s t-test was used for the statistical analysis between the two indicated populations. b Real-time PCR was performed to evaluate the expression level of miR-17 in CB CD34+ cells after transfection with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD or 17/KD1), or control (CTRL). The data are presented as the ratio of miR-17 levels (relative to U6) in 17/OE, 17/KD or 17/KD1 to that in CTRL. Each reaction was performed in triplicate. c The expression level of miR-17 in 17/OE CD34+ cells or 17/KD CD34+ cells 5 days after sorting was evaluated by real-time PCR. d The expression level of miR-17 in 17/OE CD34+CD38−/CD38+ cells or 17/KD CD34+CD38−/CD38+ cells 20 days after culture was evaluated by real-time PCR

Mentions: To determine the expression level of miR-17 in human hematopoietic cells, we first obtained two populations from human CB MNCs through the analyses of human lineage-specific CD markers. The CD34+CD38− populations expressed significantly higher levels of miR-17 in comparison to those of the CD34+CD38+ populations or MNCs (Fig. 1a). The miR-17 levels of the CD34+CD38+ populations showed a tendency, albeit insignificant, to be higher than those of the MNCs. The expression pattern of miR-17 in human hematopoietic cells is consistent with that in 32D-CSF3R cells [21]. Collectively, miR-17 levels are downregulated during the differentiation of human hematopoietic cells, which suggests that miR-17 may play a role in regulating HSC function. To analyze the function of miR-17 on primitive human hematopoietic cells, the miR-17 overexpression (17/OE) and knockdown (17/KD or 17/KD1) models were created using primary CB CD34+ cells. The expression levels of miR-17 were up- or downregulated in 17/OE and 17/KD, respectively (Fig. 1b) compared to those in CTRL. The levels of overexpression or knockdown 5 days or 20 days after culture in 17/OE CD34+CD38−/CD38+ cells or 17/KD CD34+CD38−/CD38+ cells were also checked, which maintained similar modulation (Fig. 1c and d). Based on shRNA influence on miR-17 expression in CB CD34+ cells, 17/KD was chosen for further studies.Fig. 1


miR-17 promotes expansion and adhesion of human cord blood CD34(+) cells in vitro.

Yang Y, Wang S, Miao Z, Ma W, Zhang Y, Su L, Hu M, Zou J, Yin Y, Luo J - Stem Cell Res Ther (2015)

The expression of miR-17 in CB hematopoietic CD34+CD38−/CD38+ cells. a The expression level of miR-17 in CB CD34+CD38−/CD38+ cells was evaluated by real-time PCR. Each reaction was performed in triplicate. Student’s t-test was used for the statistical analysis between the two indicated populations. b Real-time PCR was performed to evaluate the expression level of miR-17 in CB CD34+ cells after transfection with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD or 17/KD1), or control (CTRL). The data are presented as the ratio of miR-17 levels (relative to U6) in 17/OE, 17/KD or 17/KD1 to that in CTRL. Each reaction was performed in triplicate. c The expression level of miR-17 in 17/OE CD34+ cells or 17/KD CD34+ cells 5 days after sorting was evaluated by real-time PCR. d The expression level of miR-17 in 17/OE CD34+CD38−/CD38+ cells or 17/KD CD34+CD38−/CD38+ cells 20 days after culture was evaluated by real-time PCR
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4562375&req=5

Fig1: The expression of miR-17 in CB hematopoietic CD34+CD38−/CD38+ cells. a The expression level of miR-17 in CB CD34+CD38−/CD38+ cells was evaluated by real-time PCR. Each reaction was performed in triplicate. Student’s t-test was used for the statistical analysis between the two indicated populations. b Real-time PCR was performed to evaluate the expression level of miR-17 in CB CD34+ cells after transfection with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD or 17/KD1), or control (CTRL). The data are presented as the ratio of miR-17 levels (relative to U6) in 17/OE, 17/KD or 17/KD1 to that in CTRL. Each reaction was performed in triplicate. c The expression level of miR-17 in 17/OE CD34+ cells or 17/KD CD34+ cells 5 days after sorting was evaluated by real-time PCR. d The expression level of miR-17 in 17/OE CD34+CD38−/CD38+ cells or 17/KD CD34+CD38−/CD38+ cells 20 days after culture was evaluated by real-time PCR
Mentions: To determine the expression level of miR-17 in human hematopoietic cells, we first obtained two populations from human CB MNCs through the analyses of human lineage-specific CD markers. The CD34+CD38− populations expressed significantly higher levels of miR-17 in comparison to those of the CD34+CD38+ populations or MNCs (Fig. 1a). The miR-17 levels of the CD34+CD38+ populations showed a tendency, albeit insignificant, to be higher than those of the MNCs. The expression pattern of miR-17 in human hematopoietic cells is consistent with that in 32D-CSF3R cells [21]. Collectively, miR-17 levels are downregulated during the differentiation of human hematopoietic cells, which suggests that miR-17 may play a role in regulating HSC function. To analyze the function of miR-17 on primitive human hematopoietic cells, the miR-17 overexpression (17/OE) and knockdown (17/KD or 17/KD1) models were created using primary CB CD34+ cells. The expression levels of miR-17 were up- or downregulated in 17/OE and 17/KD, respectively (Fig. 1b) compared to those in CTRL. The levels of overexpression or knockdown 5 days or 20 days after culture in 17/OE CD34+CD38−/CD38+ cells or 17/KD CD34+CD38−/CD38+ cells were also checked, which maintained similar modulation (Fig. 1c and d). Based on shRNA influence on miR-17 expression in CB CD34+ cells, 17/KD was chosen for further studies.Fig. 1

Bottom Line: However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells.The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical & Research Technology, School of Medicine, University of Maryland, Baltimore, MD, 21201, USA. yangyx@bjmu.edu.cn.

ABSTRACT

Introduction: We have recently found that miR-17 is necessary in the cell-extrinsic control of cord blood (CB) CD34(+) cell function. Here, we demonstrated that the proper level of miR-17 is also necessary in the cell-intrinsic control of the hematopoietic properties of CB CD34(+) cells.

Methods: The miR-17 overexpression and knockdown models were created using primary CB CD34(+) cells transfected by the indicated vectors. Long-term culture, colony forming, adhesion and trans-well migration assays were carried out to investigate the function of miR-17 on CB CD34(+) cells in vitro. NOD prkdc (scid) Il2rg () mice were used in a SCID repopulating cell assay to investigate the function of miR-17 on CB CD34(+) cells in vivo. A two-tailed Student's t-test was used for statistical comparisons.

Results: In vitro assays revealed that ectopic expression of miR-17 promoted long-term expansion, especially in the colony-forming of CB CD34(+) cells and CD34(+)CD38(-) cells. Conversely, downregulation of miR-17 inhibited the expansion of CB CD34(+) cells. However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells. The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.

Conclusion: We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

No MeSH data available.


Related in: MedlinePlus