The different intra- and extracellular constituents of the hematopoietic stem cell (HSC) niche in the human bone marrow are tightly regulated and of momentous importance for various properties of HSCs. Some of these are regulated through β1-Integrins (CD29) which therefore dramatically influence HSC and mesenchymal stromal cell (MSC) interaction in the niche. Important regulators within these cells are microRNAs (miRNAs). These small, non-coding RNAs control the expression of around two-thirds of the human protein-coding genes. One of these miRNAs, miR-134, previously referred to be a “brain-specific” miRNA was shown to be highly expressed in MSCs in tissue-studies conducted by our group. Since the central nervous system was recently shown to be closely connected to the regulation of HSCs and MSCs, we asked whether miR-134 which has several conserved binding seed-match sequences within the 3'UTR of β1-Integrin, regulates MSC mediated properties in the bone marrow niche.
Screening of human MSC cell lines (n=4) by western blotting revealed highest β1-Integrin expression in SCP-1 cells. Transfection of SCP-1 with either siRNA directed against β1-Integrin (siCD29) or pre-miRNA-134 (pre134) revealed a downregulation of β1-Integrin at the mRNA level only in siRNA transfected cells, p=0.01. In contrast, at the protein level, as measured by western blot and FACS analysis, p=0.002, β1-Integrin was downregulated by siCD29 as well as by pre134, indicating a miRNA-specific action of repression. Confirmatory, the 3'UTR of β1-Integrin, which contains several putative binding sites for miR-134, was cloned into a pMiRReporter vector and luciferase activity was measured after cotransfection with pre134. The luciferase activity was significantly reduced in pre134 transfected cells [1.80 ± 0.46 (preCo) vs. 0.99 ± 0.49 (pre134); p<0.001]. To evaluate whether pre134 mediated reduction of β1-Integrin can modulate the adhesion potential of SCP-1, atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) was performed. Indeed, transfection of SCP-1 with siCD29 or pre134 resulted in a significantly reduced adherence as compared to their respective controls, p<0.001 and p<0.01. Furthermore, using AFM-based SCFS we investigated the interaction between 32D-cells, which have a high surface expression of the natural interaction partner of β1-Integrin VCAM-1, and SCP-1 cells. Here again, we were able to show, that 32D show a significantly lower adhesion potential to siCD29 and pre134-transfected SCP-1, p<0.001 and p<0.001, respectively. In a translational approach MSCs from healthy bone marrow donors (n=30) and from MDS patients (n=17) were screened for miRNA-expression. This analysis revealed 50% higher miR-134 transcript levels in MSCs from MDS patients [0.0057 ± 0.0021 (healthy) vs. 0.0127 ± 0.0045 (MDS); p<0.001], suggesting a potential role of this miRNA in regulating its MSC adhesion.
Regulation of adhesion of MSCs and to MSCs is important for various components of the bone marrow niche. Here, we demonstrate for the first time that β1-Integrin mediated adhesion of MSCs themselves and other cell types onto MSCs via β1-Integrin receptors can be inhibited via miR-134 overexpression. Furthermore, this newly characterized mechanism provides evidence for a potential anti-adhesive influence of miR-134. While this might not only influence adhesion, other mechanisms such as homing of HSCs as well as other cell types, might be affected by modification of miR-134 expression in the stromal niche.
Platzbecker:Amgen: Consultancy; GlaxoSmithKline: Consultancy; Celgene: Consultancy; Novartis: Consultancy.
Asterisk with author names denotes non-ASH members.