Int J Stem Cells.  2018 Nov;11(2):187-195. 10.15283/ijsc18057.

MiR-9 Controls Chemotactic Activity of Cord Blood CD34⁺ Cells by Repressing CXCR4 Expression

Affiliations
  • 1Soonchunhyang Institute of Medi-bio Science (SIMS), Soon Chun Hyang University, Cheonan, Korea. leeman@sch.ac.kr, yunklee@sch.ac.kr, yshwang0428@sch.ac.kr
  • 2Department of Obstetrics and Gynecology, Soon Chun Hyang University College of Medicine, Bucheon, Korea.
  • 3Department of Obstetrics and Gynecology, Soon Chun Hyang University Cheonan Hospital, Cheonan, Korea.
  • 4Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, USA.

Abstract

Improved approaches for promoting umbilical cord blood (CB) hematopoietic stem cell (HSC) homing are clinically important to enhance engraftment of CB-HSCs. Clinical transplantation of CB-HSCs is used to treat a wide range of disorders. However, an improved understanding of HSC chemotaxis is needed for facilitation of the engraftment process. We found that ectopic overexpression of miR-9 and antisense-miR-9 respectively down- and up-regulated C-X-C chemokine receptor type 4 (CXCR4) expression in CB-CD34⁺ cells as well as in 293T and TF-1 cell lines. Since CXCR4 is a specific receptor for the stromal cell derived factor-1 (SDF-1) chemotactic factor, we investigated whether sense miR-9 and antisense miR-9 influenced CXCR4-mediated chemotactic mobility of primary CB CD34⁺ cells and TF-1 cells. Ectopic overexpression of sense miR-9 and antisense miR-9 respectively down- and up-regulated SDF-1-mediated chemotactic cell mobility. To our knowledge, this study is the first to report that miR-9 may play a role in regulating CXCR4 expression and SDF-1-mediated chemotactic activity of CB CD34⁺ cells.

Keyword

microRNA; CXCR4; Cord blood; Cell migration

MeSH Terms

Cell Line
Cell Movement
Chemotaxis
Fetal Blood*
Hematopoietic Stem Cells
MicroRNAs
Stromal Cells
MicroRNAs

Figure

  • Fig. 1 Increase in CXCR4 expression is related to decrease in miR-9 expression in CB-CD34+ cultures. (a) CXCR4 and actin expression levels in uncultured CB-CD34+ (lane 1), from 6 hours to 4 days (lanes 2 to 5) were assessed by western blotting (WB). Actin was used as a loading control. (b) miR-9 expression levels at defined times in CB-CD34+ cultures for 4 days as analyzed by real-time PCR. The relative expression value of miR-9 was normalized to the level of U6 mRNA. Values represent mean±SE; Student’s t-test; *p<0.05 and **p<0.01. All p-values represent the significance of miR-9 expression between 0 hour and the indicated times.

  • Fig. 2 miR-9 regulation of post-transcriptional CXCR4 levels. (a) Top lines depict the sequence of miR-9 predicted to have a binding site within the CXCR4 3′UTR, middle lines depict the sequence of miR-9, and bottom lines depict the sequence of the mutant CXCR4 3′ UTR. (b) 293T cells were transfected with either luciferase-reporter constructs alone (psi-CHECK) or luciferase-reporter constructs containing WT or mutant 3′UTR of CXCR4 in the presence or absence of miR-9. The effect of miR-9 on relative luciferase activities is presented as a histogram. Values represent mean±SE. Student’s t-test: **p<0.01 and ***p<0.001. All p-values represent the significance between psi-Check of luciferase activity and the treatments.

  • Fig. 3 miR-9 regulates CXCR4 expression. (a) CB-CD34+ cells were transduced with either miR-9 or anti-miR-9, as well as with control construct. Relative miR-9 expression levels measured by real-time PCR are displayed as histograms. Relative expression values of miR-9 are calculated by normalizing them to levels of U6 mRNA. Values represent mean±SEM. Student’s t-test: *p<0.05 and ***p<0.001. (b-d) The indicated cells were transduced with either miR-9 or anti-miR-9 as well as with the control construct. Green fluorescence indicates that transduction with miR-9 and anti-miR-9 was successful. Scale bar denotes 100 μm. The expression levels of CXCR4 in the cell lines were compared by western blotting.

  • Fig. 4 miR-9 suppresses cell migration by inhibiting CXCR4. (a) The migratory cells (lower chamber) were separately harvested for the in vitro cell migration assay. In a migration assay using the Transwell system, SDF-1, which is a CXCR4 ligand, was placed in the bottom chamber of the well and 105 cells were added to the upper chamber and incubated for 24 hours. Percentage migration was calculated as (number of migrated cells/input cell number) × 100. Average cell number and standard deviation were determined from quadruplicate experiments. (b) Migration of CB-CD34+ with either miR-9 or anti-miR-9 was analyzed in the bottom chamber. Data and standard deviation were calculated from quadruplicate experiments. Student’s t-test: **p<0.01 and ***p<0.001. (c) Migration of TF-1 with either miR-9 or anti-miR-9 was analyzed in the bottom chamber. Data and standard deviation were calculated from quadruplicate experiments. Student’s t-test: *p<0.05, **p<0.01 and ***p<0.001.


Reference

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