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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 adhesion molecule expression on CB CD34+ cells after miR-17 overexpression. a The expression of N-cadherin and β1-integrin on CB CD34+ cells after miR-17 overexpressing (17/OE) or control (CTRL) cells was analyzed by flow cytometry (left panels). The results are expressed as mean ± SD from multiple independent experiments (right panels). b Flow cytometry analysis of the expression of α4-integrin, α5-integrin, CD44 and CXCR4 on miR-17 overexpressing CB CD34+ cells (green and blue lines) and CTRL CD34+ cells (red line)
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Fig4: The adhesion molecule expression on CB CD34+ cells after miR-17 overexpression. a The expression of N-cadherin and β1-integrin on CB CD34+ cells after miR-17 overexpressing (17/OE) or control (CTRL) cells was analyzed by flow cytometry (left panels). The results are expressed as mean ± SD from multiple independent experiments (right panels). b Flow cytometry analysis of the expression of α4-integrin, α5-integrin, CD44 and CXCR4 on miR-17 overexpressing CB CD34+ cells (green and blue lines) and CTRL CD34+ cells (red line)

Mentions: Ectopic miR-17 promotes the expansion of CB CD34+ cells in vitro but the hematopoietic reconstitution capability of 17/OE cells is reduced in vivo, which displays inconsistency. Except for expansion, the hematopoietic reconstitution capability of engrafted hematopoietic cells is also affected by the components from a physiological niche where HSCs reside in vivo. Successful hematopoietic reconstitution of human hematopoietic cells in engrafted mice needs proper proliferation, suitable attachment between HSCs and the bone marrow niche, as well as migration of HSCs in vivo. To examine whether the reduced hematopoietic reconstitution capability of CB CD34+ cells upon miR-17 modulation in vivo is a result of improper attachment or migration in engrafted mice, we examined the expression patterns of selected adhesion and homing molecules known to be important for the hematopoietic reconstitution of human HSCs in the engrafted mice. As shown in Fig. 4a, 17/OE cells showed significantly higher expression levels of N-cadherin (3.16 ± 0.82-fold, n = 4) as well as β1-integrin (3.30 ± 0.98-fold, n = 4) compared to CTRL cells. We also analyzed the expression of α4 and α5 integrins in 17/OE cells, which were shown to be slightly increased. The expression levels of CD44 and CXCR4 were almost unchanged upon ectopic miR-17 (Fig. 4b). The improper expression of N-cadherin and β1-integrin on CD34+ cells upon miR-17 overexpression raised the possibility that the adhesion between 17/OE cells and their niche in vivo is regulated abnormally, which further leads to the reduced hematopoietic reconstitution capability of 17/OE cells in vivo. After knockdown of miR-17, there was a trend towards a decrease in the expression of β1-integrin and N-cadherin (Additional file 2: Figure S2) , although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05).Fig. 4


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 adhesion molecule expression on CB CD34+ cells after miR-17 overexpression. a The expression of N-cadherin and β1-integrin on CB CD34+ cells after miR-17 overexpressing (17/OE) or control (CTRL) cells was analyzed by flow cytometry (left panels). The results are expressed as mean ± SD from multiple independent experiments (right panels). b Flow cytometry analysis of the expression of α4-integrin, α5-integrin, CD44 and CXCR4 on miR-17 overexpressing CB CD34+ cells (green and blue lines) and CTRL CD34+ cells (red line)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4562375&req=5

Fig4: The adhesion molecule expression on CB CD34+ cells after miR-17 overexpression. a The expression of N-cadherin and β1-integrin on CB CD34+ cells after miR-17 overexpressing (17/OE) or control (CTRL) cells was analyzed by flow cytometry (left panels). The results are expressed as mean ± SD from multiple independent experiments (right panels). b Flow cytometry analysis of the expression of α4-integrin, α5-integrin, CD44 and CXCR4 on miR-17 overexpressing CB CD34+ cells (green and blue lines) and CTRL CD34+ cells (red line)
Mentions: Ectopic miR-17 promotes the expansion of CB CD34+ cells in vitro but the hematopoietic reconstitution capability of 17/OE cells is reduced in vivo, which displays inconsistency. Except for expansion, the hematopoietic reconstitution capability of engrafted hematopoietic cells is also affected by the components from a physiological niche where HSCs reside in vivo. Successful hematopoietic reconstitution of human hematopoietic cells in engrafted mice needs proper proliferation, suitable attachment between HSCs and the bone marrow niche, as well as migration of HSCs in vivo. To examine whether the reduced hematopoietic reconstitution capability of CB CD34+ cells upon miR-17 modulation in vivo is a result of improper attachment or migration in engrafted mice, we examined the expression patterns of selected adhesion and homing molecules known to be important for the hematopoietic reconstitution of human HSCs in the engrafted mice. As shown in Fig. 4a, 17/OE cells showed significantly higher expression levels of N-cadherin (3.16 ± 0.82-fold, n = 4) as well as β1-integrin (3.30 ± 0.98-fold, n = 4) compared to CTRL cells. We also analyzed the expression of α4 and α5 integrins in 17/OE cells, which were shown to be slightly increased. The expression levels of CD44 and CXCR4 were almost unchanged upon ectopic miR-17 (Fig. 4b). The improper expression of N-cadherin and β1-integrin on CD34+ cells upon miR-17 overexpression raised the possibility that the adhesion between 17/OE cells and their niche in vivo is regulated abnormally, which further leads to the reduced hematopoietic reconstitution capability of 17/OE cells in vivo. After knockdown of miR-17, there was a trend towards a decrease in the expression of β1-integrin and N-cadherin (Additional file 2: Figure S2) , although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05).Fig. 4

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