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MiR-193a-5p has been observed to have oncogenic or tumor suppressive functions in different kinds of cancers, but its role and molecular mechanism in osteosarcoma are elusive. Na+/Ca2+ exchangers (NCX1, NCX2 and NCX3) normally extrude Ca2+ from the cell, and deregulation of the intracellular Ca2+ homeostasis is related to several kinds of diseases, including cancer. The present study demonstrated that miR-193a-5p was upregulated in osteosarcoma tissues compared with the corresponding adjacent noncancerous tissues, and promoted colony formation, migration, invasion and epithelial-mesenchymal transition (EMT) in osteosarcoma cells (SaOS-2 and U-2OS), as well as metastasis in a murine xenograft model. Tandem mass tag-based quantitative proteomics analysis identified NCX2 as a potential target of miR-193a-5p. Luciferase activity assays and Western blotting further confirmed that miR-193a-5p recognized the 3′-untranslated region of NCX2 mRNA, and negatively regulated NCX2 expression. NCX2 was downregulated in osteosarcoma tissues, and its expression was negatively correlated with miR-193a-5p levels. Ectopic expression of NCX2 in osteosarcoma cells could reverse the oncogenicity of miR-193a-5p, indicating that miR-193a-5p exerted its effects by targeting NCX2. Further study demonstrated that NCX2 suppresses Ca2+-dependent Akt phosphorylation by decreasing intracellular Ca2+ concentration, then inhibited EMT process. Treatment with the antagomir against miR-193a-5p sensitized osteosarcoma to the Akt inhibitor afuresertib in a murine xenograft model. In conclusion, a miR-193a-5p/NCX2/AKT signaling axis contributes to the progression of osteosarcoma, which may provide a new therapeutic target for osteosarcoma treatment.

Spinal muscular atrophy (SMA) is a severe neuromuscular disease affecting infants caused by alterations of the survival motor neuron gene, which results in progressive degeneration of motor neurons (MNs). Although an effective treatment for SMA patients has been recently developed, the molecular pathway involved in selective MN degeneration has not been yet elucidated. In particular, miR-206 has been demonstrated to play a relevant role in the regeneration of neuromuscular junction in several MN diseases, and particularly it is upregulated in the quadriceps, tibialis anterior, spinal cord, and serum of SMA mice. In the present paper, we demonstrated that miR-206 was transiently upregulated also in the brainstem of the mouse model of SMA, SMAΔ7, in the early phase of the disease paralleling MN degeneration and was down-regulated in the late symptomatic phase. To prevent this downregulation, we intracerebroventricularly injected miR-206 in SMA pups, demonstrating that miR-206 reduced the severity of SMA pathology, slowing down disease progression, increasing survival rate, and improving behavioral performance of mice. Interestingly, exogenous miRNA-206-induced upregulation caused a reduction of the predicted target sodium calcium exchanger isoform 2, NCX2, one of the main regulators of intracellular [Ca2+] and [Na+]. Therefore, we hypothesized that miR-206 might exert part of its neuroprotective effect modulating NCX2 expression in SMA disease. Motor neuron degeneration occurs very early in the facial nuclei of SMA mice, paralleling a transient upregulation of miR-206. The prolonged increase of miR-206 induced by i.c.v. administration reduces motor neuron loss, ameliorates behavioral performance, and increases mouse lifespan, also through the modulation of the plasma membrane Na+/Ca2+ exchanger 2, NCX2.

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN) is a frequent adverse event of colorectal cancer treatment. OIPN encompasses a chronic and an acute syndrome. The latter consists of transient axonal hyperexcitability, due to unbalance in Na+ voltage-operated channels (Na+VOC). This leads to sustained depolarisation which can activate the reverse mode of the Na+/Ca2+ exchanger 2 (NCX2), resulting in toxic Ca2+ accumulation and axonal damage (ADa). We explored the role of NCX2 in in vitro and in vivo settings. Embryonic rat Dorsal Root Ganglia (DRG) organotypic cultures treated with SEA0400 (SEA), a NCX inhibitor, were used to assess neuroprotection in a proof-of-concept and pilot study to exploit NCX modulation to prevent ADa. In vivo, OHP treated mice (7 mg/Kg, i.v., once a week for 8 weeks) were compared with a vehicle-treated group (n = 12 each). Neurophysiological and behavioural testing were performed to characterise acute and chronic OIPN, and morphological analyses were performed to detect ADa. Immunohistochemistry, immunofluorescence, and western blotting (WB) analyses were also performed to demonstrate changes in NCX2 immunoreactivity and protein expression. In vitro, NCX inhibition was matched by ADa mitigation. In the in vivo part, after verifyingboth acute and chronic OIPN had ensued, we confirmed via immunohistochemistry, immunofluorescence, and WB that a significant NCX2 alteration had ensued in the OHP group. Our data suggest NCX2 involvement in ADa development, paving the way to a new line of research to prevent OIPN.

An exceptionally low calcium (Ca2+) concentration in the inner ear endolymph ([Ca2+]endolymph) is crucial for proper auditory and vestibular function. The endolymphatic sac (ES) is believed to critically contribute to the maintenance of this low [Ca2+]endolymph. Here, we investigated the immunohistochemical localization of proteins that are presumably involved in the sensing and transport of extracellular Ca2+ in the murine ES epithelium. Light microscopic and fluorescence immunolabeling in paraffin-embedded murine ES tissue sections (male C57BL/6 mice, 6–8 weeks old) demonstrated the presence of the calcium-sensing receptor CaSR, transient receptor potential cation channel subtypes TRPV5 and TRPV6, sarco/endoplasmic reticulum Ca2+-ATPases SERCA1 and SERCA2, Na+/Ca2+ exchanger NCX2, and plasma membrane Ca2+ ATPases PMCA1 and PMCA4 in ES epithelial cells. These proteins exhibited (i) membranous (apical or basolateral) or cytoplasmic localization patterns, (ii) a proximal-to-distal labeling gradient within the ES, and (iii) different distribution patterns among ES epithelial cell types (mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs)). Notably, in the inner ear membranous labyrinth, CaSR was exclusively localized in MRCs, suggesting a unique role of the ES epithelium in CaSR-mediated sensing and control of [Ca2+]endolymph. Structural loss of the distal ES, which is consistently observed in Meniere’s disease, may therefore critically disturb [Ca2+]endolymph and contribute to the pathogenesis of Meniere’s disease. Electronic supplementary material The online version of this article (10.1007/s00441-019-03062-2) contains supplementary material, which is available to authorized users.

Dysregulation of sodium [Na+]iand calcium [Ca2+]ihomeostasis plays a pivotal role in the pathophysiology of cerebral ischemia. Three gene products of the sodium–calcium exchanger family NCX1, NCX2, and NCX3 couple, in a bidirectional way, the movement of these ions across the cell membrane during cerebral ischemia. Each isoform displays a selective distribution in the rat brain. To determine whether NCX gene expression can be regulated after cerebral ischemia, we used NCX isoform-specific antisense radiolabeled probes to analyze, by radioactive in situ hybridization histochemistry, the pattern of NCX1, NCX2, and NCX3 transcripts in the ischemic core, periinfarct area, as well as in nonischemic brain regions, after 6 and 24 h of permanent middle cerebral artery occlusion (pMCAO) in rats. We found that in the focal region, comprising divisions of the prefrontal, somatosensory, and insular cortices, all three NCX transcripts were downregulated. In the periinfarct area, comprising part of the motor cortex and the lateral compartments of the caudate-putamen, NCX2 messenger ribonucleic acid (mRNA) was downregulated, whereas NCX3 mRNA was significantly upregulated. In remote nonischemic brain regions such as the prelimbic and infralimbic cortices, and tenia tecta, both NCX1 and NCX3 transcripts were upregulated, whereas in the medial caudate-putamen only NCX3 transcripts increased. In all these intact regions, NCX2 signal strongly decreased. These results indicate that NCX gene expression is regulated after pMCAO in a differential manner, depending on the exchanger isoform and region involved in the insult. These data may provide a better understanding of each NCX subtype's pathophysiologic role and may allow researchers to design appropriate pharmacological strategies to treat brain ischemia.