👤 Simon J O'Carroll

The highly conserved long non-coding RNA (lncRNA) MIR505HG has been primarily recognized as a precursor for microRNAs (miR)-424 and miR-503. However, studies have since demonstrated that MIR503HG has distinct functions from its associated miRNAs, playing important roles in cell proliferation, invasion, apoptosis, and differentiation. While these miRNAs are known to influence cardiomyocyte differentiation, the specific role of MIR503HG in heart development remains unexplored. We seek to determine how MIR503HG deletion impacts ventricular chamber development and to identify underlying molecular mechanisms. To study the role of the lncRNA in vivo, we generated a functional MIR503HG knockout mouse model (MIR503HG-/-) using a synthetic polyadenylation signal to terminate MIR503HG transcription without affecting miR-424/503 expression. We performed morphological analyses on embryonic and adult hearts using microCT along with cardiac functional analysis via transthoracic echocardiography. We further apply single-nuclei RNA sequencing (snRNA-seq) on adult hearts to identify potential molecular mechanisms underlying the observed phenotypes. Functional deletion of MIR503HG alone was associated with reduced compact myocardium thickness and increased trabecular myocardium in the left ventricle (LV) at embryonic day 17.5 compared to wild-type mice, indicating a LV non-compaction (LVNC) phenotype. Moreover, adult MIR503HG-/- mutant hearts showed increased trabecular complexity, impaired LV relaxation, and mitral valve regurgitation. SnRNA-seq further revealed altered expression of several genes associated with cardiomyocyte function and LVNC, including Actc1, Mib1, Mybpc3, and Myh7. Lastly, Notch1 activity was also significantly increased in mutant hearts which has been previously associated with LVNC. MIR503HG plays a role in ventricular chamber development, and its deletion leads to an LVNC phenotype independent of the miRNA cluster within its locus, highlighting its importance in cardiac development and disease. We further suggest that abnormal Notch1 activity may underpin the LVNC phenotype presented. Show less

We have recently demonstrated that invasive melanoma cells are capable of disrupting the brain endothelial barrier integrity. This was shown using ECIS biosensor technology, which revealed rapid disruption via the paracellular junctions. In this paper, we demonstrate that melanoma cells secrete factors (e.g., cytokines) that weaken the endothelial barrier integrity. Through proteome profiling, we attempt to identify the barrier-disrupting cytokines. Melanoma conditioned media were collected from three New Zealand melanoma lines. ECIS technology was used to assess if the conditioned media disrupted the endothelial barrier independent of the melanoma cells. The melanoma cell secretome was assessed using cytometric bead array (CBA), Luminex immunoassay and multiplex Proteome Profilers, to detect the expression of secretory proteins, which may facilitate metastasis. Finally, ECIS technology was used to assess the direct effects of secreted proteins identified as candidates from the proteome screens. We show that melanoma-conditioned media significantly disrupted the brain endothelial barrier, however, to a much lesser extent than the cells from which they were collected. Cytokine and proteome profiling of the conditioned media showed evidence of high concentrations of approximately 15 secreted proteins (including osteopontin, IL-8, GDF-15, MIF and VEGF). These 15 secreted proteins were expressed variably across the melanoma lines. Surprisingly, the addition of these individually to the brain endothelial cells did not substantially affect the barrier integrity. ANGPTL-4 and TGFβ were also produced by the melanoma cells. Whilst TGFβ-1 had a pronounced effect on the barrier integrity, surprisingly ANGPTL-4 did not. However, its C-terminal fragment did and within a very similar period to the conditioned media, albeit not to the same extent. Herein we show that melanoma cells produce a wide-range of soluble factors at high concentrations, which most likely favour support or survival of the cancer cells. Most of these, except for TGFβ-1 and the C-terminal fragment of ANGPTL-4, did not have an impact on the integrity of the brain endothelial cells. Show less