hsa-mir-30c promotes the invasive phenotype of metastatic breast cancer cells by targeting NOV/CCN3

BACKGROUND: For treatment and prevention of metastatic disease, one of the premier challenges is the identification of pathways and proteins to target for clinical intervention. Micro RNAs (miRNAs) are short, non-coding RNAs, which regulate cellular activities by either mRNA degradation or translational inhibition. Our studies focused on the invasive properties of hsa-mir30c based on its high expression in MDA-MB-231 metastatic cells and our bioinformatic analysis of the Cancer Genome Atlas that identified aberrant hsa-mir-30c to be associated with poor survival. METHODS: Contributions of hsa-mir-30c to breast cancer cell invasion were examined by Matrigel invasion transwell assays following modulation of hsa-mir-30c or hsa-mir-30c* levels in MDA-MB-231 cells. hsa-mir-30c in silico predicted targets linked to cell invasion were screened for targeting by hsa-mir-30c in metastatic breast cancer cells by RT-qPCR. The contribution to invasion by a target of hsa-mir-30c, Nephroblastoma overexpressed (NOV), was characterized by siRNA and invasion assays. Significant effects were determined using Student\u27s T-tests with Welch\u27s correction for unequal variance. RESULTS: MCF-7 and MDA-MB-231 cells were used as models of poorly invasive and late-stage metastatic disease, respectively. By modulating the levels of hsa-mir-30c in these cells, we observed concomitant changes in breast cancer cell invasiveness. From predicted targets of hsa-mir-30c that were related to cellular migration and invasion, NOV/CCN3 was identified as a novel target of hsa-mir-30c. Depleting NOV by siRNA caused a significant increase in the invasiveness of MDA-MB-231 cells is a regulatory protein associated with the extracellular matrix. CONCLUSIONS: NOV/CCN3 expression, which protects cells from invasion, is known in patient tumors to inversely correlate with advanced breast cancer and metastasis. This study has identified a novel target of hsa-mir-30c, NOV, which is an inhibitor of the invasiveness of metastatic breast cancer cells. Thus, hsa-mir-30c-mediated inhibition of NOV levels promotes the invasive phenotype of MDA-MB-231 cells and significantly, the miR-30/NOV pathways is independent of RUNX2, a known target of hsa-mir-30c that promotes osteolytic disease in metastatic breast cancer cells. Our findings allow for mechanistic insight into the clinical observation of poor survival of patients with elevated hsa-mir-30c levels, which can be considered for miRNA-based translational studies

MicroRNA Levels as Prognostic Markers for the Differentiation Potential of Human Mesenchymal Stromal Cell Donors

The ability of human mesenchymal stromal/stem cells (hMSCs) to differentiate into various mesenchymal cell lineages makes them a promising cell source for the use in tissue repair strategies. Because the differentiation potential of hMSCs differs between donors, it is necessary to establish biomarkers for the identification of donors with high differentiation potential. Here, we show that microRNA (miRNA) expression levels are effective for distinguishing donors with high differentiation potential from low differentiation potential. Twenty human MSC donors were initially tested for marker expression and differentiation potential. In particular, chondrogenic differentiation potential was evaluated on the basis of histological matrix formation, mRNA expression levels of chondrogenic marker genes, and quantitative glycosaminoglycan deposition. Three donors out of twenty were identified as donors with high chondrogenic potential, whereas nine showed moderate and eight low chondrogenic potential. Expression profiles of miRNAs involved in chondrogenesis and cartilage homeostasis were used for the distinction between high-performance hMSCs and low-performance hMSCs. Global mRNA expression profiles of the donors before the onset of chondrogenic differentiation revealed minor differences in gene expression between low and high chondrogenic performers. However, analysis of miRNA expression during a seven-day differentiation period identified miR-210 and miR-630 as positive regulators of chondrogenesis. In contrast, miR-181 and miR-34a, which are negative regulators of chondrogenesis, were upregulated during differentiation in low performing donors. In conclusion, profiling of hMSC donors for a specific panel of miRNAs may have prognostic value for selecting donors with high differentiation potential to improve hMSC-based strategies for tissue regeneration

Wnt signaling in triple-negative breast cancer

Wnt signaling regulates a variety of cellular processes, including cell fate, differentiation, proliferation and stem cell pluripotency. Aberrant Wnt signaling is a hallmark of many cancers. An aggressive subtype of breast cancer, known as triple-negative breast cancer (TNBC), demonstrates dysregulation in canonical and non-canonical Wnt signaling. In this review, we summarize regulators of canonical and non-canonical Wnt signaling, as well as Wnt signaling dysfunction that mediates the progression of TNBC. We review the complex molecular nature of TNBC and the emerging therapies that are currently under investigation for the treatment of this disease

Snorc is a novel cartilage specific small membrane proteoglycan expressed in differentiating and articular chondrocytes

Objective: Maintenance of chondrocyte phenotype is a major issue in prevention of degeneration and repair of articular cartilage. Although the critical pathways in chondrocyte maturation and homeostasis have been revealed, the in-depth understanding is deficient and novel modifying components and interaction partners are still likely to be discovered. Our focus in this study was to characterize a novel cartilage specific gene that was identified in mouse limb cartilage during embryonic development. Methods: Open access bioinformatics tools and databases were used to characterize the gene, predicted protein and orthologs in vertebrate species. Immunohistochemistry and mRNA expression methodology were used to study tissue specific expression. Fracture callus and limb bud micromass culture were utilized to study the effects of BMP-2 during experimental chondrogenesis. Fusion protein with C-terminal HA-tag was expressed in Cos7 cells, and the cell lysate was studied for putative glycosaminoglycan attachment by digestion with chondroitinase ABC and Western blotting. Results: The predicted molecule is a small, 121 amino acids long type I single-pass transmembrane chondroitin sulfate proteoglycan, that contains ER signal peptide, lumenal/extracellular domain with several threonines/serines prone to O-N-acetylgalactosamine modification, and a cytoplasmic tail with a Yin-Yang site prone to phosphorylation or O-N-acetylglucosamine modification. It is highly conserved in mammals with orthologs in all vertebrate subgroups. Cartilage specific expression was highest in proliferating and prehypertrophic zones during development, and in adult articular cartilage, expression was restricted to the uncalcified zone, including chondrocyte clusters in human osteoarthritic cartilage. Studies with experimental chondrogenesis models demonstrated similar expression profiles with Sox9, Acan and Col2a1 and up-regulation by BMP-2. Based on its cartilage specific expression, the molecule was named Snorc, (Small NOvel Rich in Cartilage). Conclusion: A novel cartilage specific molecule was identified which marks the differentiating chondrocytes and adult articular chondrocytes with possible functions associated with development and maintenance of chondrocyte phenotype

Runx1 is associated with breast cancer progression in MMTV-PyMT transgenic mice and its depletion in vitro inhibits migration and invasion

Runx1 is a transcription factor essential for definitive hematopoiesis, and genetic abnormalities in Runx1 cause leukemia. Runx1 is functionally promiscuous and acts as either an oncogene or tumor suppressor gene in certain epithelial cancers. Recent evidence suggests that Runx1 is an important factor in breast cancer, however its role remains ambiguous. Here, we addressed whether Runx1 has a specific pathological role during breast cancer progression and show that Runx1 has an oncogenic function. We observed elevated Runx1 expression in a subset of human breast cancers. Furthermore, throughout the course of disease progression in a classical mouse model of breast cancer (i.e., the MMTV-PyMT transgenic model), Runx1 expression increases in the primary site (mammary gland) and is further upregulated in tumors and distal lung metastatic lesions. Ex vivo studies using tumor epithelial cells derived from these mice express significantly higher levels of Runx1 than normal mammary epithelial cells. The tumor cells exhibit increased rates of migration and invasion, indicative of an aggressive cancer phenotype. Inhibition of Runx1 expression using RNA interference significantly abrogates these cancer-relevant phenotypic characteristics. Importantly, our data establish that Runx1 contributes to murine mammary tumor development and malignancy and potentially represents a key disease-promoting and prognostic factor in human breast cancer progression and metastasis. This article is protected by copyright. All rights reserved

MicroRNA Functions in Osteogenesis and Dysfunctions in Osteoporosis

MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression that control osteoblast mediated bone formation and osteoclast-related bone remodeling. Deregulation of miRNA mediated mechanisms is emerging as an important pathological factor in bone degeneration (eg, osteoporosis) and other bone-related diseases. MiRNAs are intriguing regulatory molecules that are networked with cell signaling pathways and intricate transcriptional programs through ingenuous circuits with remarkably simple logic. This overview examines key principles by which miRNAs control differentiation of osteoblasts as they evolve from mesenchymal stromal cells during osteogenesis, or of osteoclasts as they originate from monocytic precursors in the hematopoietic lineage during osteoclastogenesis. Of particular note are miRNAs that are temporally upregulated during osteoblastogenesis (eg, miR-218) or osteoclastogenesis (eg, miR-148a). Each miRNA stimulates differentiation by suppressing inhibitory signaling pathways ('double-negative' regulation). The excitement surrounding miRNAs in bone biology stems from the prominent effects that individual miRNAs can have on biological transitions during differentiation of skeletal cells and correlations of miRNA dysfunction with bone diseases. MiRNAs have significant clinical potential which is reflected by their versatility as disease-specific biomarkers and their promise as therapeutic agents to ameliorate or reverse bone tissue degeneration