1Goethe-Universität Institut für Kardiovaskuläre Physiologie Frankfurt, Deutschland; 2Max-Planck-Institute for Heart- and Lung Research Bad Nauheim, Deutschland; 3Universitätsklinikum Frankfurt Hämatologie und internistische Onkologie Frankfurt, Deutschland
Motivation: SMANTIS is a physiologically important lncRNA maintaining endothelial cell function and inhibiting cell adhesion to monocytes. However, expression profiling of lncRNA SMANTIS showed a much higher expression in peripheral blood mononuclear cells (PBMCs). Interestingly, lncRNA SMANTIS was upregulated in monocytes differentiated from inducible pluripotent stem cells. Therefore, we aim to functionally characterize SMANTIS in monocytes in respect to its cardiovascular and disease-related function.
Methods/Results: During the differentiation of inducible pluripotent stem cells to monocytes, SMANTIS expression strongly appeared with the differentiation to monocytes and was lost once monocytes differentiated further into macrophages. RNA-Seq of a (>100) patient cohort with acute myeloid leukemia (AML) displayed a reduction of SMANTIS with the degree of AML differentiation. Given the fact that monocytes are an important part of the innate immune system initiating an inflammatory response, we stimulated THP-1 cells, with and without CRISPR/Cas9-mediated deletion of SMANTIS, with lipopolysaccharide (LPS). Although there was no significant change in the immune response, the deletion of SMANTIS favored significantly the attachment to unstimulated endothelial cells and inflammatory conditions amplified the adhesion of SMANTIS deleted THP-1 to endothelial cells. RNA-Seq additionally showed differentially expressed genes associated with cell adhesion supporting the hypothesis that SMANTIS might be involved in the regulation of monocyte adhesion to endothelial cells. VCAN and ITGAM were under the top ten upregulated genes involved in cell adhesion. Furthermore, genes associated with osteoclast differentiation were identified. Since ITGAM is a marker of osteoclast precursors, THP-1 were differentiated into osteoclast-like cells, which revealed that osteoclast marker genes were higher expressed in SMANTIS-deleted THP-1. Interestingly, SMANTIS interacted with the transcription factor RUNX1, a frequent mutational target in AML and regulator of osteoclast differentiation, in an RNA antisense-pulldown. Subsequently, CUT&RUN for RUNX1 revealed around 800 downregulated peaks after SMANTIS deletion, these included peaks in RELB and OCSTAMP.
Conclusion: These data indicate a diverse function of lncRNA SMANTIS in the physiological role of monocytes apart from the inflammatory response. SMANTIS interaction with RUNX1 is especially interesting for AML, hematopoietic-, and osteoclast-differentiation and might imply an attractive target for anti-inflammatory therapy.