Regulatory Potential of miRNAs on CXCL12 Expression in Human Mesenchymal Stem Cells (MSC)

Abstract 1332

CXCL12 is the major CXC chemokine produced by various cell types of the hematopoietic niche. It is well known for it‘s regulatory potential in physiological processes like stem cell mobilization and homing. In addition it participates in regulation of proliferation and survival of cells with expression of it‘s receptor CXCR4. Thus, variable CXCL12 levels can influence a multitude of mechanisms associated with stem cell migration, homing and proliferation, e.g. during stem cell mobilisation CXCL12 levels are down regulated in MSC and osteoblasts thereby enabling conversion of CD34 positive cells into the circulation. The regulatory mechanisms of this process are however incompletely understood. In a systematic approach we investigated whether miRNAs are able to mediate CXCL12 regulation in human MSC. We transfected the immortalized human MSC cell line SCP-1 with a library consisting of 702 different pre-miRNAs. Subsequently, CXCL12 level determination by ELISA revealed a total of 27 miRNAs downregulating CXCL12 and 34 miRNAs which upregulated CXCL-12. Since the most important effect of miRNAs is the interaction with the 3 ‘UTR of target genes we further validated our candidate miRNAs with a luciferase assay using the 3 ‘UTR of CXCL12 transcript variant 2. Here, 8 miRNAs (miR-23, 130b, 135a, 200b, 200 c, 216, 222 and 602) were shown to interact significantly with CXCL12. Next, expression of candidate miRNAs was determined in 46 human resting primary MSC samples which proved strongest expression of miR-23 in MSC, miR-130b and 222 are expressed at lower but detectable levels, whereas the other candidate miRNAs showed no expression in human MSC. Subsequently, transfection of pre-miR-23 molecules in primary human MSC reduced both CXCL12 protein values and the attraction of migrating cells in a transwell assay.

In conclusion, using a large scale library transfection approach we identified miR-23 as an important regulator of CXCL-12 In human MSC.