Toward the blood-borne miRNome of human diseases.

In a multicenter study, we determined the expression profiles of 863 microRNAs by array analysis of 454 blood samples from human individuals with different cancers or noncancer diseases, and validated this 'miRNome' by quantitative real-time PCR. We detected consistently deregulated profiles for all tested diseases; pathway analysis confirmed disease association of the respective microRNAs. We observed significant correlations (P = 0.004) between the genomic location of disease-associated genetic variants and deregulated microRNAs.

Impaired TGF-ß induced growth inhibition contributes to the increased proliferation rate of neural stem cells harboring mutant p53.

Gliomas have been classified according to their histological properties. However, their respective cells of origin are still unknown. Neural progenitor cells (NPC) from the subventricular zone (SVZ) can initiate tumors in murine models of glioma and are likely cells of origin in the human disease. In both, p53 signaling is often functionally impaired which may contribute to tumor formation. Also, TGF-beta, which under physiological conditions exerts a strong control on the proliferation of NPCs in the SVZ, is a potent mitogen on glioma cells. Here, we approach on the crosstalk between p53 and TGF-beta by loss of function experiments using NPCs derived from p53 mutant mice, as well as pharmacological inhibition of TGF-beta signaling using TGF-beta receptor inhibitors. NPC derived from p53 mutant mice showed increased clonogenicity and more rapid proliferation than their wildtype counterparts. Further, NPC derived from p53(mut/mut) mice were insensitive to TGF-beta induced growth arrest. Still, the canonical TGF-beta signaling pathway remained functional in the absence of p53 signaling and expression of key proteins as well as phosphorylation and nuclear translocation of SMAD2 were unaltered. TGF-beta-induced p21 expression could, in contrast, only be detected in p53(wt/wt) but not in p53(mut/mut) NPC. Conversely, inhibition of TGF-beta signaling using SB431542 increased proliferation of p53(wt/wt) but not of p53(mut/mut) NPC. In conclusion, our data suggest that the TGF-beta induced growth arrest in NPC depends on functional p53. Mutational inactivation of p53 hence contributes to increased proliferation of NPC and likely to the formation of hyperplasia of the SVZ observed in p53 deficient mice in vivo.

Whole miRNome-wide differential co-expression of microRNAs.

Co-regulation of genes has been extensively analyzed, however, rather limited knowledge is available on co-regulations within the miRNome. We investigated differential co-expression of microRNAs (miRNAs) based on miRNome profiles of whole blood from 540 individuals. These include patients suffering from different cancer and non-cancer diseases, and unaffected controls. Using hierarchical clustering, we found 9 significant clusters of co-expressed miRNAs containing 2-36 individual miRNAs. Through analyzing multiple sequencing alignments in the clusters, we found that co-expression of miRNAs is associated with both sequence similarity and genomic co-localization. We calculated correlations for all 371,953 pairs of miRNAs for all 540 individuals and identified 184 pairs of miRNAs with high correlation values. Out of these 184 pairs of miRNAs, 16 pairs (8.7%) were differentially co-expressed in unaffected controls, cancer patients and patients with non-cancer diseases. By computing correlated and anti-correlated miRNA pairs, we constructed a network with 184 putative co-regulations as edges and 100 miRNAs as nodes. Thereby, we detected specific clusters of miRNAs with high and low correlation values. Our approach represents the most comprehensive co-regulation analysis based on whole miRNome-wide expression profiling. Our findings further decrypt the interactions of miRNAs in normal and human pathological processes.

Shigella mediated depletion of macrophages in a murine breast cancer model is associated with tumor regression.

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TDeltaaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TDeltaaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression.These data support TAMs as useful targets for antitumor therapy and highlight attenuated bacterial pathogens as potential tools.

The non-classical MHC molecule HLA-G protects human muscle cells from immune-mediated lysis: implications for myoblast transplantation and gene therapy.

HLA-G is a non-classical MHC class I molecule with highly limited tissue distribution which has been attributed chiefly immune-regulatory functions. We previously have reported that HLA-G is expressed in inflamed muscle in vivo and by cultured myoblasts in vitro. Here, we used the in vitro models of human myoblasts or TE671 muscle rhabdomyosarcoma cells to characterize the functional role of HLA-G for muscle immune cell interactions. Gene transfer of the two major isoforms of HLA-G (transmembranous HLA-G1 and soluble HLA-G5) into TE671 rendered these cells resistant to alloreactive lysis by direct inhibition of natural killer (NK) cells, and CD4 and CD8 T cells. Further, HLA-G reduced alloproliferation, interfered with effective priming of antigen-specific cytotoxic T cells and reduced antigen-specific alloreactive lysis. HLA-G pre-induced on cultured myoblasts inhibited lysis by alloreactive peripheral blood mononuclear cells. This protection was reversed by a neutralizing HLA-G antibody. Interestingly, a few HLA-G-positive cells within a population of HLA-G-negative muscle target cells conveyed significant inhibitory effects on alloreactive lysis. Our results reveal further insights into the immunobiology of muscle and suggest that ectopic expression of HLA-G may promote the survival of transplanted myoblasts in the future treatment of hereditary muscle diseases. Further, HLA-G could represent a novel self-derived anti-inflammatory principle applicable in strategies against inflammatory aggression.