The chemokine CXCL12 regulates the interaction between hematopoietic stem and progenitor cells and bone marrow stromal cells. Although its relevance in the bone marrow niche is well recognized, the regulation of CXCL12 by microRNA is not completely understood. We transfected a library of 486 microRNA in the bone marrow stromal cell line SCP-1 and studied the expression of CXCL12. Twenty-seven microRNA were shown to downregulate expression of CXCL12. Eight microRNA (miR-23a, 130b, 135, 200b, 200c, 216, 222, and 602) interacted directly with the 3´UTR of CXCL12. Next, we determined that only miR-23a is predicted to bind to the 3´UTR and is strongly expressed in primary bone marrow stromal cells. Modulation of miR-23a changes the migratory potential of hematopoietic progenitor cells in co-culture experiments. We discovered that TGFB1 mediates its inhibitory effect on CXCL12 levels by upregulation of miR-23a. This process was partly reversed by miR-23a molecules. Finally, we determined an inverse expression of CXCL12 and miR-23a in stromal cells from patients with myelodysplastic syndrome indicating that the interaction has a pathophysiological role. Here, we show for the first time that CXCL12-targeting miR23a regulates the functional properties of the hematopoietic niche. MicroRNA-23a mediates post-transcriptional regulation of CXCL12 in bone marrow stromal cells ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o nproteases. 11,12 Furthermore, CXCL12 expression can be transcriptionally modulated by a variety of cytokines and growth factors, namely transforming growth factor-beta 1 (TGFB1). TGFB1 has a negative growth effect on HSPC which is mediated, in part, by the regulation of CXCL12 arising from stromal cells. 13 Thus, engraftment and response to cytotoxic drugs may vary according to CXCL12 levels provided by niche cells. 14,15 Recently, Pillai et al. suggested that CXCL12 can also be regulated by microRNA (miRNA) in human marrow stromal cells. 16 There are more than 1000 miRNA which form 1-2% of the human genome. Approximately half of human structural genes are predicted to be under miRNA control. In animals, miRNA act by targeting the 3ú ntranslated region (UTR) of genes with the consequence of repressing output of the respective protein. A classical switch interaction is used to avoid protein expression in a particular cell type, whereas tuning interactions are needed to supply the cell with the optimal level of protein. 17 In the hematopoietic system, CXCL12 must be fine-tuned in response to differing physiological requirements.We, therefore, decided to study the interaction of CXCL12 and miRNA by applying a strategy of overexpression of a library to reveal the various miRNA responsible for CXCL12 regulation in primary human bone marrow stromal cells (hBMSC). Moreover, we mapped expression of candidate miRNA in primary hBMSC to understand their physiological function in fine tuning CXCL12 expression in the hematopoietic niche. Methods Cell linesHS5, HS27, HL60, K562, KG1a, and HeLa cell lines were ob...

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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. Disclosures: No relevant conflicts of interest to declare.