miR-126-3p Promotes Matrix-Dependent Perivascular Cell Attachment, Migration and Intercellular Interaction.

microRNAs (miRNAs) can regulate the interplay between perivascular cells (PVC) and endothelial cells (EC) during angiogenesis, but the relevant PVC-specific miRNAs are not yet defined. Here, we identified miR-126-3p and miR-146a to be exclusively upregulated in PVC upon interaction with EC, determined their influence on the PVC phenotype and elucidate their molecular mechanisms of action. Specifically the increase of miR-126-3p strongly promoted the motility of PVC on the basement membrane-like composite and stabilized networks of EC. Subsequent miRNA target analysis showed that miR-126-3p inhibits SPRED1 and PLK2 expression, induces ERK1/2 phosphorylation and stimulates TLR3 expression to modulate cell-cell and cell-matrix contacts of PVC. Gain of expression experiments in vivo demonstrated that miR-126-3p stimulates PVC coverage of newly formed vessels and transform immature into mature, less permeable vessels. In conclusion we showed that miR-126-3p regulates matrix-dependent PVC migration and intercellular interaction to modulate vascular integrity. Stem Cells 2016;34:1297-1309.

Genome-wide profiling of microRNAs in adipose mesenchymal stem cell differentiation and mouse models of obesity.

In recent years, there has been accumulating evidence that microRNAs are key regulator molecules of gene expression. The cellular processes that are regulated by microRNAs include e.g. cell proliferation, programmed cell death and cell differentiation. Adipocyte differentiation is a highly regulated cellular process for which several important regulating factors have been discovered, but still not all are known to fully understand the underlying mechanisms. In the present study, we analyzed the expression of 597 microRNAs during the differentiation of mouse mesenchymal stem cells into terminally differentiated adipocytes by real-time RT-PCR. In total, 66 miRNAs were differentially expressed in mesenchymal stem cell-derived adipocytes compared to the undifferentiated progenitor cells. To further study the regulation of these 66 miRNAs in white adipose tissue in vivo and their dependence on PPARγ activity, mouse models of genetically or diet induced obesity as well as a mouse line expressing a dominant negative PPARγ mutant were employed.

Analysis of putative miRNA binding sites and mRNA 3' ends as targets for siRNA-mediated gene knockdown.

Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that generate highly functional siRNAs for gene knockdown. Nevertheless, "difficult-to-silence" target messenger RNAs (mRNAs) still exist for which no functionally validated siRNAs are available. MicroRNA (miRNA) sites in the mRNA 3'UTR, which interact with miRNA-loaded RNA-induced silencing complex (miRISC) for posttranscriptional gene regulation, provide alternative potentially accessible sites for siRNA. To investigate this, we designed siRNAs directed against single putative miRNA sites (misiRNAs) as predicted by miRNA target databases as well as siRNAs against other regions within the 3'UTR of "difficult-to-silence" targets in this proof-of-principle study. Although their design was not fully optimized, the misiRNAs generally caused higher knockdown than previously designed siRNAs for these targets. In general, knockdown by misiRNAs targeting the miRNA seed region was specific for the target mRNA, and misiRNAs targeting 1 nt upstream of miRNA seed region were similarly potent. We also systematically screened the 3'UTR of two mRNA targets using siRNA-tiling experiments. 5' and 3' regions of the p21-activated kinase 6 (PAK6) 3'UTR were found accessible, whereas the middle portion was largely inaccessible for siRNA knockdown. In ribosomal protein S6 kinase (RPS6KB1) 3'UTR, however, only the 5' region was accessible for siRNA knockdown. Detailed analysis of 10 further "difficult-to-silence" targets revealed that siRNA accessibility at the mRNA 3' end is not a general phenomenon, at least in "difficult-to-silence" targets, as we could not detect significant knockdown by siRNAs directed against this region.

Enrichment strategies for siRNA-transfected cells in an untransfected background.

Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present proof-of-concept data from two different strategies for enrichment of siRNA-transfected cells. In the first approach, a heterologous surface antigen is expressed from a plasmid that is co-transfected with an siRNA targeting an endogenous mRNA. The surface antigen is then used for enrichment of successfully transfected cells using antibody-coated magnetic particles. In the second strategy, a eukaryotic antibiotic resistance gene is expressed from a co-transfected plasmid. Addition of the corresponding antibiotic 24h after transfection results in killing of untransfected cells, which can be washed away. Elimination of untransfected cells will allow more accurate interpretation of the effects of gene silencing.

An siRNA-based system for differential regulation of ectopic gene expression constructs.

We describe an siRNA (short interfering RNA)-based approach for temporary and reversible regulation of engineered expression constructs in cells. Control of cloned genes can be achieved by cotransfection of unique siRNAs, complementary to artificial target sequences, which are integral parts of an expression vector. Application of this method allows simultaneous or mutual-differential regulation of two or more gene constructs within the same cell, reducing unwanted side effects. This method provides several advantages over promoter regulatory systems employing chemical compounds.

RNA interference: PCR strategies for the quantification of stable degradation-fragments derived from siRNA-targeted mRNAs.

mRNA targeted by siRNA is endogeneously cleaved into a 5'- and a 3'-fragment and finally degraded in cells. Little is known about the relative stability and degradation kinetics of these 5'- and 3'-fragments after the siRNA mediated first cut. We present a qRT-PCR protocol which allows the determination of the optimal time point for mRNA analyses, helping to avoid the generation of false positive effects in downstream experiments, such as microarray analysis, which may be caused by undegraded fragments of a siRNA-targeted mRNA.

Molecular cloning and expression analysis of a novel member of the Disintegrin and Metalloprotease-Domain (ADAM) family.

We cloned and characterized the cDNA and the expression pattern of a novel member of the murine 'Disintegrin and Metalloprotease-Domain Family' (ADAM). The predicted protein sequence reveals highest homology to the testase-subgroup, composed of ADAM 24, ADAM 25 and ADAM 26 and is therefore called testase 4. Reverse transcription-polymerase chain reaction showed a strong expression of testase 4 in the adult testis, but not in any other organ or embryonic stage tested. Careful characterization by in situ hybridization confirmed specific expression of testase 4 in maturing sperm cells of 6-week-old mice, whereas no specific hybridization pattern was detectable in testes of 2.5-week-old mice. These data indicate a correlation between testase 4 expression and spermatogenesis and/or fertilization.

c-Cbl binding and ubiquitin-dependent lysosomal degradation of membrane-associated Notch1.

Regulation of Notch1 activity is critical for cell fate decisions and differentiation of skeletal myoblasts. We have employed the skeletal myoblast cell line C2C12 to study posttranslational regulation of Notch1 protein levels during myogenesis. Although the major degradation pathway of the activated intracellular Notch1 fragment appears to involve ubiquitination and degradation by the 26 S proteasome, we provide evidence for an alternative catalytic pathway where the endogenous, transmembrane form of Notch1 is targeted to the lysosomal compartment. Immunoprecipitation analysis revealed ubiquitin-dependent accumulation of transmembrane Notch1 protein after treatment with the lysosomal inhibitor chloroquine but not after treatment with various proteasome inhibitors. This finding was supported by the observation that the transmembrane form of Notch1 was tyrosine-phosphorylated and specifically coprecipitated with the ubiquitin ligase c-Cbl. Our data suggest a regulatory mechanism down-regulating Notch1 protein levels already at the cellular surface, possibly with consequences for Notch-dependent signal transduction during terminal differentiation processes.