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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

Effects of miR-17 overexpression on adhesion and migration of CB CD34+ cells. a The adhesion of miR-17 overexpressing (17/OE) CB CD34+ cells to N-cadherin or vascular cell adhesion molecule-1 (VCAM1) was significantly increased compared with that of control (CRTL) cells. Results are shown as mean + SD (n = 8 for N-cadherin and n = 10 for VCAM1). *p < 0.05, between 17/OE and CTRL groups (Student’s t-test). b The adhesion of 17/OE CD34+ cells with β1-integrin deficient (β1/KD) to VCAM1 was significantly decreased compared with that of CRTL cells. Mean + SD is shown (*p < 0.05, Student’s t-test). The CTRL is 17/OE CD34+ cells with scrambled shRNA. c The specific adhesion of CD34+ cells to N-cadherin or VCAM1 was confirmed by antibody blocking experiments. The adhesion was measured as described above. The adhesion of pre-treated cells was significant inhibited compared with that of non-treated cells (*p < 0.05, Student’s t-test). d The migration of 17/OE CB CD34+ cells towards SDF1α was slightly increased compared with that of CTRL cells, but statistical analyses indicated it did not meet statistical significance (p > 0.3, Student’s t-test)
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Fig5: Effects of miR-17 overexpression on adhesion and migration of CB CD34+ cells. a The adhesion of miR-17 overexpressing (17/OE) CB CD34+ cells to N-cadherin or vascular cell adhesion molecule-1 (VCAM1) was significantly increased compared with that of control (CRTL) cells. Results are shown as mean + SD (n = 8 for N-cadherin and n = 10 for VCAM1). *p < 0.05, between 17/OE and CTRL groups (Student’s t-test). b The adhesion of 17/OE CD34+ cells with β1-integrin deficient (β1/KD) to VCAM1 was significantly decreased compared with that of CRTL cells. Mean + SD is shown (*p < 0.05, Student’s t-test). The CTRL is 17/OE CD34+ cells with scrambled shRNA. c The specific adhesion of CD34+ cells to N-cadherin or VCAM1 was confirmed by antibody blocking experiments. The adhesion was measured as described above. The adhesion of pre-treated cells was significant inhibited compared with that of non-treated cells (*p < 0.05, Student’s t-test). d The migration of 17/OE CB CD34+ cells towards SDF1α was slightly increased compared with that of CTRL cells, but statistical analyses indicated it did not meet statistical significance (p > 0.3, Student’s t-test)

Mentions: Through adhesion to the corresponding components from niche in vivo, the adhesive moleculars expressed by hematopoietic cells regulated the interaction between hematopoietic cells and their niche. To examine whether the reduced hematopoietic reconstitution ability of 17/OE cells is related to the upregulated expression of N-cadherin and β1-integrin, we performed the adhesion assays in vitro. After incubation with the coated ligands, CB CD34+ cells showed a significant increase in the adhesion to N-cadherin or VCAM1 upon ectopic miR-17 according to the CFC output (Fig. 5a). In order to identify whether the increase in adhesion potential of 17/OE cells to VCAM1 in vitro is directly from the altered expression levels of β1-integrin in 17/OE cells, we further knocked down the expression of β1-integrin in 17/OE cells with β1-integrin-specific shRNA (here called β1/KD; Fig. 5b). The adhesion potential of 17/OE CD34+ cells to VCAM1 was significantly blocked upon β1-integrin knockdown (Fig. 5b), which suggested that β1-integrin expressed on 17/OE CD34+ cells mediated, at least in part, the increase in interaction between 17/OE CD34+ cells and VCAM1 caused by ectopic miR-17. In order to demonstrate that the observed adhesion between 17/OE cells and N-cadherin or VCAM1 was directly mediated by their corresponding ligands, the cells were pre-incubated with soluble anti-N-cadherin peptide or anti-CD29 antibody, respectively, prior to performing adhesive assay. The adhesion potential was determined as described above. As shown in Fig. 5c, the specific adhesion of pre-incubated cells to each ligand was significantly reduced compared with that of nontreated cells. It is of note that the migration of miR-17 overexpressed CB CD34+ cells towards SDF1α was slightly decreased compared to that of control cells. However, statistical analyses of this cohort indicated that it did not meet statistical significance (Fig. 5d), suggesting that the reduced hematopoietic reconstitution capability of miR-17 overexpressed CB CD34+ cells in vivo does not result from the migration defect of transplanted 17/OE cells.Fig. 5


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)

Effects of miR-17 overexpression on adhesion and migration of CB CD34+ cells. a The adhesion of miR-17 overexpressing (17/OE) CB CD34+ cells to N-cadherin or vascular cell adhesion molecule-1 (VCAM1) was significantly increased compared with that of control (CRTL) cells. Results are shown as mean + SD (n = 8 for N-cadherin and n = 10 for VCAM1). *p < 0.05, between 17/OE and CTRL groups (Student’s t-test). b The adhesion of 17/OE CD34+ cells with β1-integrin deficient (β1/KD) to VCAM1 was significantly decreased compared with that of CRTL cells. Mean + SD is shown (*p < 0.05, Student’s t-test). The CTRL is 17/OE CD34+ cells with scrambled shRNA. c The specific adhesion of CD34+ cells to N-cadherin or VCAM1 was confirmed by antibody blocking experiments. The adhesion was measured as described above. The adhesion of pre-treated cells was significant inhibited compared with that of non-treated cells (*p < 0.05, Student’s t-test). d The migration of 17/OE CB CD34+ cells towards SDF1α was slightly increased compared with that of CTRL cells, but statistical analyses indicated it did not meet statistical significance (p > 0.3, Student’s t-test)
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Fig5: Effects of miR-17 overexpression on adhesion and migration of CB CD34+ cells. a The adhesion of miR-17 overexpressing (17/OE) CB CD34+ cells to N-cadherin or vascular cell adhesion molecule-1 (VCAM1) was significantly increased compared with that of control (CRTL) cells. Results are shown as mean + SD (n = 8 for N-cadherin and n = 10 for VCAM1). *p < 0.05, between 17/OE and CTRL groups (Student’s t-test). b The adhesion of 17/OE CD34+ cells with β1-integrin deficient (β1/KD) to VCAM1 was significantly decreased compared with that of CRTL cells. Mean + SD is shown (*p < 0.05, Student’s t-test). The CTRL is 17/OE CD34+ cells with scrambled shRNA. c The specific adhesion of CD34+ cells to N-cadherin or VCAM1 was confirmed by antibody blocking experiments. The adhesion was measured as described above. The adhesion of pre-treated cells was significant inhibited compared with that of non-treated cells (*p < 0.05, Student’s t-test). d The migration of 17/OE CB CD34+ cells towards SDF1α was slightly increased compared with that of CTRL cells, but statistical analyses indicated it did not meet statistical significance (p > 0.3, Student’s t-test)
Mentions: Through adhesion to the corresponding components from niche in vivo, the adhesive moleculars expressed by hematopoietic cells regulated the interaction between hematopoietic cells and their niche. To examine whether the reduced hematopoietic reconstitution ability of 17/OE cells is related to the upregulated expression of N-cadherin and β1-integrin, we performed the adhesion assays in vitro. After incubation with the coated ligands, CB CD34+ cells showed a significant increase in the adhesion to N-cadherin or VCAM1 upon ectopic miR-17 according to the CFC output (Fig. 5a). In order to identify whether the increase in adhesion potential of 17/OE cells to VCAM1 in vitro is directly from the altered expression levels of β1-integrin in 17/OE cells, we further knocked down the expression of β1-integrin in 17/OE cells with β1-integrin-specific shRNA (here called β1/KD; Fig. 5b). The adhesion potential of 17/OE CD34+ cells to VCAM1 was significantly blocked upon β1-integrin knockdown (Fig. 5b), which suggested that β1-integrin expressed on 17/OE CD34+ cells mediated, at least in part, the increase in interaction between 17/OE CD34+ cells and VCAM1 caused by ectopic miR-17. In order to demonstrate that the observed adhesion between 17/OE cells and N-cadherin or VCAM1 was directly mediated by their corresponding ligands, the cells were pre-incubated with soluble anti-N-cadherin peptide or anti-CD29 antibody, respectively, prior to performing adhesive assay. The adhesion potential was determined as described above. As shown in Fig. 5c, the specific adhesion of pre-incubated cells to each ligand was significantly reduced compared with that of nontreated cells. It is of note that the migration of miR-17 overexpressed CB CD34+ cells towards SDF1α was slightly decreased compared to that of control cells. However, statistical analyses of this cohort indicated that it did not meet statistical significance (Fig. 5d), suggesting that the reduced hematopoietic reconstitution capability of miR-17 overexpressed CB CD34+ cells in vivo does not result from the migration defect of transplanted 17/OE cells.Fig. 5

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