Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/-) mice.

Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn (+/-), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in mdx/utrn (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca2+ homeostasis and abrogate tissue fibrosis in DMD.

UTRN as a potential biomarker in breast cancer: a comprehensive bioinformatics and in vitro study.

Utrophin (UTRN), known as a tumor suppressor, potentially regulates tumor development and the immune microenvironment. However, its impact on breast cancer's development and treatment remains unstudied. We conducted a thorough examination of UTRN using both bioinformatic and in vitro experiments in this study. We discovered UTRN expression decreased in breast cancer compared to standard samples. High UTRN expression correlated with better prognosis. Drug sensitivity tests and RT-qPCR assays revealed UTRN's pivotal role in tamoxifen resistance. Furthermore, the Kruskal-Wallis rank test indicated UTRN's potential as a valuable diagnostic biomarker for breast cancer and its utility in detecting T stage of breast cancer. Additionally, our results demonstrated UTRN's close association with immune cells, inhibitors, stimulators, receptors, and chemokines in breast cancer (BRCA). This research provides a novel perspective on UTRN's role in breast cancer's prognostic and therapeutic value. Low UTRN expression may contribute to tamoxifen resistance and a poor prognosis. Specifically, UTRN can improve clinical decision-making and raise the diagnosis accuracy of breast cancer.

circ_UTRN inhibits ferroptosis of ARJ21 cells to attenuate acute pancreatitis progression by regulating the miR-760-3p/FOXO1/GPX4 axis.

Aim: To explore the function of circ_UTRN in acute pancreatitis (AP). Methods: After exposing AR42J cells to caerulein, the levels of circ_UTRN, miR-760-3p, and glutathione peroxidase 4 (GPX4) were determined by quantitative polymerase chain reaction. Additionally, GPX4 and forkhead box O1 (FOXO1) protein levels were assessed by western blot. The levels of oxidative stress and ferroptosis in the supernatant of the treated AR42J cells were also assessed using commercial kits. Results: circ_UTRN inhibited caerulein-induced oxidative stress and ferroptosis by binding with miR-760-3p. Additionally, miR-760-3p directly targeted FOXO1, thereby regulating GPX4 levels. Furthermore, GPX4 knockdown abolished the effect of miR-760-3p downregulation in AP. Conclusion: circ_UTRN inhibited oxidative stress and ferroptosis by regulating the miR-760-3p/FOXO1/GPX4 axis. This is a potential new treatment strategy for AP.

A Protocol for Simultaneous In Vivo Imaging of Cardiac and Neuroinflammation in Dystrophin-Deficient MDX Mice Using [18F]FEPPA PET.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by dystrophin loss-notably within muscles and the central neurons system. DMD presents as cognitive weakness, progressive skeletal and cardiac muscle degeneration until pre-mature death from cardiac or respiratory failure. Innovative therapies have improved life expectancy; however, this is accompanied by increased late-onset heart failure and emergent cognitive degeneration. Thus, better assessment of dystrophic heart and brain pathophysiology is needed. Chronic inflammation is strongly associated with skeletal and cardiac muscle degeneration; however, neuroinflammation's role is largely unknown in DMD despite being prevalent in other neurodegenerative diseases. Here, we present an inflammatory marker translocator protein (TSPO) positron emission tomography (PET) protocol for in vivo concomitant assessment of immune cell response in hearts and brains of a dystrophin-deficient mouse model [mdx:utrn(+/-)]. Preliminary analysis of whole-body PET imaging using the TSPO radiotracer, [18F]FEPPA in four mdx:utrn(+/-) and six wildtype mice are presented with ex vivo TSPO-immunofluorescence tissue staining. The mdx:utrn(+/-) mice showed significant elevations in heart and brain [18F]FEPPA activity, which correlated with increased ex vivo fluorescence intensity, highlighting the potential of TSPO-PET to simultaneously assess presence of cardiac and neuroinflammation in dystrophic heart and brain, as well as in several organs within a DMD model.

Circ_UTRN ameliorates caerulein-induced acute pancreatitis in vitro via reducing inflammation and promoting apoptosis through miR-320-3p/PTK2 axis.

OBJECTIVES: Circular RNAs (circRNAs) have been demonstrated to play important roles in acute pancreatitis (AP). Herein, this study aimed to investigate the role and mechanism of circRNAs utrophin (circ_UTRN) in AP. METHODS: In vitro cultured rat pancreatic acinar cell line AR42J was exposed to caerulein (10 nmol/L) to mimic an AP cell model. The levels of circ_UTRN and microRNA (miR)-320-3p and protein tyrosine kinase 2 (PTK2) were examined using quantitative real-time polymerase chain reaction and Western blot assays. Cell apoptosis was analysed by flow cytometry and Western blot assays. ELISA was employed to detect the levels of tumour necrosis factor-α (TNF-α), IL-1ß and IL-6. The binding interaction between miR-320-3p and circ_UTRN or PTK2 was verified using dual-luciferase reporter assay. KEY FINDINGS: The expression of circ_UTRN was decreased by caerulein in pancreatic acinar cells, ectopic overexpression of circ_UTRN reduced inflammation and promoted apoptosis in caerulein-mediated pancreatic acinar cells. In a mechanical study, circ_UTRN served as a sponge of miR-320-3p, and miR-320-3p directly targeted PTK2. Rescue assay suggested that the promotion of apoptosis and inhibition of inflammation induced by circ_UTRN re-expression in caerulein-mediated pancreatic acinar cells were partially abolished by miR-320-3p overexpression or PTK2 knockdown. Besides that, miR-320-3p inhibition impaired caerulein-induced cell apoptosis arrest and inflammation via targeting PTK2. CONCLUSIONS: Up-regulation of circ_UTRN in pancreatic acinar cells attenuates caerulein-evoked cell apoptosis arrest and inflammation enhancement via miR-320-3p/PTK2, suggesting that circ_UTRN/miR-320-3p/PTK2 axis might be engaged in caerulein-induced AP.

UTRN inhibits melanoma growth by suppressing p38 and JNK/c-Jun signaling pathways.

BACKGROUND: Utrophin (UTRN), as a tumor suppressor gene, is involved in various cancer progression. The function of UTRN in the melanoma process and the related molecular mechanisms are still unclear. Herein, we studied the function of UTRN in melanoma growth and the relevant molecular mechanisms. METHODS: Using the GEO database and UCSC Xena project, we compared the expression of UTRN in non-cancerous and melanoma tissues. Immunohistochemistry (IHC) staining, qRT-PCR and Western Blot (WB) were performed to evaluate UTRN expression in clinical samples. A total of 447 cases with UTRN expression data, patient characteristics and survival data were extracted from TCGA database and analyzed. After stable transduction and single cell cloning, the proliferation ability of A375 human melanoma cells was analyzed by Cell Counting Kit­8 (CCK) and 5­ethynyl­2'­deoxyuridine (EdU) incorporation assays. GSEA was performed to predict the mechanism by which UTRN regulated melanoma growth. Then WB analysis was used to assess the protein expression levels of pathway signaling in overexpression (EXP) melanoma cells. Epac activator 8-pCPT-2'-O-Me-cAMP was then used to evaluate the proliferation ability by activation of p38 and JNK/c-Jun signaling pathways. RESULTS: Data from GEO and UCSC Xena project indicated that UTRN expression was decreased in melanoma. Experiment on clinical samples further confirmed our finding. TCGA results showed that a reduced expression of UTRN in 447 melanoma samples was associated with advanced clinical characteristics (T stage, Clark level, ulceration), shorter survival time and poorer prognosis. In addition, up-regulated UTRN expression inhibited melanoma cell proliferation when compared to control group. MAPK signaling pathway was presented in both KEGG and BioCarta databases by using GSEA tool. WB results confirmed the down-regulated expression of p38, JNK1 and c-Jun in EXP group when compared to control group. Epac activator 8-pCPT-2'-O-Me-cAMP treatment could partially rescue proliferation of tumor cells. CONCLUSION: We have demonstrated that reduced UTRN predicted poorer prognosis and UTRN inhibited melanoma growth via p38 and JNK1/c-Jun pathways. Therefore, UTRN could serve as a tumor suppressor and novel prognostic biomarker for melanoma patients.

Genome Editing-Mediated Utrophin Upregulation in Duchenne Muscular Dystrophy Stem Cells.

Utrophin upregulation is considered a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). A number of microRNAs (miRNAs) post-transcriptionally regulate utrophin expression by binding their cognate sites in the 3' UTR. Previously we have shown that miRNA: UTRN repression can be alleviated using miRNA let-7c site blocking oligonucleotides (SBOs) to achieve utrophin upregulation and functional improvement in mdx mice. Here, we used CRISPR/Cas9-mediated genome editing to delete five miRNA binding sites (miR-150, miR-296-5p, miR-133b, let-7c, miR-196b) clustered in a 500 bp inhibitory miRNA target region (IMTR) within the UTRN 3' UTR, for achieving higher expression of endogenous utrophin. Deleting the UTRN IMTR in DMD patient-derived human induced pluripotent stem cells (DMD-hiPSCs) resulted in ca. 2-fold higher levels of utrophin protein. Differentiation of the UTRN edited DMD-hiPSCs (UTRNΔIMTR) by MyoD overexpression resulted in increased sarcolemmal α-sarcoglycan staining consistent with improved dystrophin glycoprotein complex (DGC) restoration. These results demonstrate that CRISPR/Cas9-based UTRN genome editing offers a novel utrophin upregulation therapeutic strategy applicable to all DMD patients, irrespective of the dystrophin mutation status.

A Genome-Wide Association Study Identifies UTRN Gene Polymorphism for Restless Legs Syndrome in a Korean Population.

OBJECTIVE: Restless legs syndrome (RLS) is a highly heritable and common neurological sensorimotor disease disturbing sleep. The objective of study was to investigate significant gene for RLS by performing GWA and replication study in a Korean population. METHODS: We performed a GWA study for RLS symptom group (n=325) and non-RLS group (n=2,603) from the Korea Genome Epidemiology Study. We subsequently performed a replication study in RLS and normal controls (227 RLS and 229 controls) to confirm the present GWA study findings as well as previous GWA study results. RESULTS: In the initial GWA study of RLS, we observed an association of rs11645604 (OR=1.531, p=1.18×10-6) in MPHOSPH6 on chromosome 16q23.3, rs1918752 (OR=0.6582, p=1.93×10-6) and rs9390170 (OR=0.6778, p=7.67×10-6) in UTRN on chromosome 6q24. From the replication samples, we found rs9390170 in UTRN (p=0.036) and rs3923809 and rs9296249 in BTBD9 (p=0.045, p=0.046, respectively) were significantly associated with RLS. Moreover, we found the haplotype polymorphisms of rs9357271, rs3923809, and rs9296249 (overall p=5.69×10-18) in BTBD9 was associated with RLS. CONCLUSION: From our sequential GWA and replication study, we could hypothesize rs9390170 polymorphism in UTRN is a novel genetic marker for susceptibility to RLS. Regarding with utrophin, which is encoded by UTRN, is preferentially expressed in the neuromuscular synapse and myotendinous junctions, we speculate that utrophin is involved in RLS, particularly related to the neuromuscular aspects.

A Genome-Wide Association Study Identifies UTRN Gene Polymorphism for Restless Legs Syndrome in a Korean Population

OBJECTIVE: Restless legs syndrome (RLS) is a highly heritable and common neurological sensorimotor disease disturbing sleep. The objective of study was to investigate significant gene for RLS by performing GWA and replication study in a Korean population. METHODS: We performed a GWA study for RLS symptom group (n=325) and non-RLS group (n=2,603) from the Korea Genome Epidemiology Study. We subsequently performed a replication study in RLS and normal controls (227 RLS and 229 controls) to confirm the present GWA study findings as well as previous GWA study results. RESULTS: In the initial GWA study of RLS, we observed an association of rs11645604 (OR=1.531, p=1.18×10−6) in MPHOSPH6 on chromosome 16q23.3, rs1918752 (OR=0.6582, p=1.93×10−6) and rs9390170 (OR=0.6778, p=7.67×10−6) in UTRN on chromosome 6q24. From the replication samples, we found rs9390170 in UTRN (p=0.036) and rs3923809 and rs9296249 in BTBD9 (p=0.045, p=0.046, respectively) were significantly associated with RLS. Moreover, we found the haplotype polymorphisms of rs9357271, rs3923809, and rs9296249 (overall p=5.69×10−18) in BTBD9 was associated with RLS. CONCLUSION: From our sequential GWA and replication study, we could hypothesize rs9390170 polymorphism in UTRN is a novel genetic marker for susceptibility to RLS. Regarding with utrophin, which is encoded by UTRN, is preferentially expressed in the neuromuscular synapse and myotendinous junctions, we speculate that utrophin is involved in RLS, particularly related to the neuromuscular aspects.

Application of 3-d echocardiography and gated micro-computed tomography to assess cardiomyopathy in a mouse model of duchenne muscular dystrophy.

The purpose of this study was to measure changes in cardiac function as cardiomyopathy progresses in a mouse model of Duchenne muscular dystrophy using 3-D ECG-gated echocardiography. This study is the first to correlate cardiac volumes acquired using 3-D echocardiography with those acquired using retrospectively gated micro-computed tomography (CT). Both were further compared with standard M-mode echocardiography and histologic analyses. We found that although each modality measures a decrease in cardiac function as disease progresses in mdx/utrn(-/-) mice (n = 5) compared with healthy C57BL/6 mice (n = 8), 3-D echocardiography has higher agreement with gold-standard measurements acquired by gated micro-CT, with little standard deviation between measurements. M-Mode echocardiography measurements, in comparison, exhibit considerably greater variability and user bias. Given the radiation dose associated with micro-CT and the geometric assumptions made in M-mode echocardiography to calculate ventricular volume, we suggest that use of 3-D echocardiography has important advantages that may allow for the measurement of early disease changes that occur before overt cardiomyopathy.

Mechanical and non-mechanical functions of Dystrophin can prevent cardiac abnormalities in Drosophila.

Dystrophin-deficiency causes cardiomyopathies and shortens the life expectancy of Duchenne and Becker muscular dystrophy patients. Restoring Dystrophin expression in the heart by gene transfer is a promising avenue to explore as a therapy. Truncated Dystrophin gene constructs have been engineered and shown to alleviate dystrophic skeletal muscle disease, but their potential in preventing the development of cardiomyopathy is not fully understood. In the present study, we found that either the mechanical or the signaling functions of Dystrophin were able to reduce the dilated heart phenotype of Dystrophin mutants in a Drosophila model. Our data suggest that Dystrophin retains some function in fly cardiomyocytes in the absence of a predicted mechanical link to the cytoskeleton. Interestingly, cardiac-specific manipulation of nitric oxide synthase expression also modulates cardiac function, which can in part be reversed by loss of Dystrophin function, further implying a signaling role of Dystrophin in the heart. These findings suggest that the signaling functions of Dystrophin protein are able to ameliorate the dilated cardiomyopathy, and thus might help to improve heart muscle function in micro-Dystrophin-based gene therapy approaches.

Engraftment of ES-Derived Myogenic Progenitors in a Severe Mouse Model of Muscular Dystrophy.

Controlled myogenic differentiation of mouse embryonic stem cells by Pax3 combined with purification of PDGFαR+Flk-1- paraxial mesoderm results in the efficient in vitro generation of early skeletal myogenic progenitors. Upon transplantation into dystrophin-deficient mdx mice, these progenitors promote significant regeneration that is accompanied by improvement in muscle contractility. In this study, we aimed to raise the bar and assess the therapeutic potential of these cells in a more clinically relevant model of muscular dystrophy: the dystrophin-utrophin double-knockout (dKO) mouse. Unlike mdx mice, which display a mild phenotype, dKO mice are severely ill, displaying progressive muscle wasting, impaired mobility, and premature death. Here we show that in this very severe model of DMD, transplantation of Pax3-induced ES-derived skeletal myogenic progenitors results in significant engraftment as evidenced by the presence of Dystrophin+ myofibers with restoration of ß-dystroglycan and eNOS within the sarcolemma, and enhanced strengthen of treated muscles. These findings demonstrate that ES-derived myogenic cell preparations are capable of engrafting in severely dystrophic muscle, and promote significant regeneration, providing a rationale for further studies on the potential therapeutic application of these cells in muscular dystrophies.