Inhibition of miR-144-3p/FOXO1 Attenuates Diabetic Keratopathy Via Modulating Autophagy and Apoptosis.

Purpose: Diabetic keratopathy (DK) is a vision-threatening disease that occurs in people with diabetes. Mounting evidence indicates that microRNAs (miRNAs) are indispensable in nerve regeneration within DK. Herein, the role of miRNAs associated with DK, especially focusing on autophagy and apoptosis regulation, was investigated. Methods: To identify differentially expressed miRNAs, we performed miRNA sequencing on trigeminal ganglion (TG) tissues derived from streptozotocin-induced type 1 diabetic mellitus (T1DM) and normal mice. MiR-144-3p was chosen for the subsequent experiments. To explore the regulatory role of miR-144-3p in DK, miRNA antagomir was utilized to inhibit miR-144-3p expression. Bioinformatic tools were used to predict the target genes of miR-144-3p, and a dual-luciferase reporter assay was then applied for validation. Autophagy and apoptosis activities were measured utilizing TUNEL staining, immunofluorescence staining, and Western blotting. Results: Overall, 56 differentially expressed miRNAs were detected in diabetic versus control mice. In the diabetic mouse TG tissue, miR-144-3p expression was aberrantly enhanced, whereas decreasing its expression contributed to improved diabetic corneal re-epithelialization and nerve regeneration. Fork-head Box O1 (FOXO1) was validated as a target gene of miR-144-3p. Overexpression of FOXO1 could prevent both inadequate autophagy and excessive apoptosis in DK. Consistently, a specific miR-144-3p inhibition enhanced autophagy and prevented apoptosis in DK. Conclusions: In this study, our research confirmed the target binding relationship between miR-144-3p and FOXO1. Inhibiting miR-144-3p might modulate autophagy and apoptosis, which could generate positive outcomes for corneal nerves via targeting FOXO1 in DK.

Cardiac miR-19a/19b was induced and hijacked by CVB3 to facilitate virus replication via targeting viral genomic RdRp-encoding region.

Coxsackievirus B3 (CVB3) is one of the major pathogens of viral myocarditis, lacking specific anti-virus therapeutic options. Increasing evidence has shown an important involvement of the miR-17-92 cluster both in virus infection and cardiovascular development and diseases, while its role in CVB3-induced viral myocarditis remains unclear. In this study, we found that miR-19a and miR-19b were significantly up-regulated in heart tissues of CVB3-infected mice and exerted a significant facilitatory impact on CVB3 biosynthesis and replication, with a more pronounced effect observed in miR-19b, by targeting the encoding region of viral RNA-dependent RNA polymerase 3D (RdRp, 3Dpol) to increase viral genomic RNA stability. The virus-promoting effects were nullified by the synonymous mutations in the viral 3Dpol-encoding region, which corresponded to the seed sequence shared by miR-19a and miR-19b. In parallel, treatment with miR-19b antagomir not only resulted in a noteworthy suppression of CVB3 replication and infection in infected cells, but also demonstrated a significant reduction in the cardiac viral load of CVB3-infected mice, resulting in a considerable alleviation of myocarditis. Collectively, our study showed that CVB3-induced cardiac miR-19a/19b contributed to viral myocarditis via facilitating virus biosynthesis and replication, and targeting miR-19a/19b might represent a novel therapeutic target for CVB3-induced viral myocarditis.

Emerging role of MicroRNA-Based theranostics in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), with its high mortality and short survival rate, continues to be one of the deadliest malignancies despite relentless efforts and several technological advances. The poor prognosis of HCC and the few available treatments are to blame for the low survival rate, which emphasizes the importance of creating new, effective diagnostic markers and innovative therapy strategies. In-depth research is being done on the potent biomarker miRNAs, a special class of non-coding RNA and has shown encouraging results in the early identification and treatment of HCC in order to find more viable and successful therapeutics for the disease. It is beyond dispute that miRNAs control cell differentiation, proliferation, and survival and, depending on the genes they target, can either promote tumorigenesis or suppress it. Given the vital role miRNAs play in the biological system and their potential to serve as ground-breaking treatments for HCC, more study is required to fully examine their theranostic potential.

Programming inactive RNA-binding small molecules into bioactive degraders.

Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.

miRNAs and Stem Cells as Promising Diagnostic and Therapeutic Targets for Alzheimer's Disease.

Alzheimer's disease (AD) is a cumulative progressive neurodegenerative disease characterized mainly by impairment in cognitive functions accompanied by memory loss, disturbance in behavior and personality, and difficulties in learning. Although the main causes of AD pathogenesis are not fully understood yet, amyloid-ß peptides and tau proteins are supposed to be responsible for AD onset and pathogenesis. Various demographic, genetic, and environmental risk factors are involved in AD onset and pathogenesis such as age, gender, several genes, lipids, malnutrition, and poor diet. Significant changes were observed in microRNA (miRNA) levels between normal and AD cases giving hope for a diagnostic procedure for AD through a simple blood test. As yet, only two classes of AD therapeutic drugs are approved by FDA. They are classified as acetylcholinesterase inhibitors and N-methyl-D-aspartate antagonists (NMDA). Unfortunately, they can only treat the symptoms but cannot cure AD or stop its progression. New therapeutic approaches were developed for AD treatment including acitretin due to its ability to cross blood-brain barrier in the brain of rats and mice and induce the expression of ADAM 10 gene, the α-secretase of human amyloid-ß protein precursor, stimulating the non-amyloidogenic pathway for amyloid-ß protein precursor processing resulting in amyloid-ß reduction. Also stem cells may have a crucial role in AD treatment as they can improve cognitive functions and memory in AD rats through regeneration of damaged neurons. This review spotlights on promising diagnostic techniques such as miRNAs and therapeutic approaches such as acitretin and/or stem cells keeping in consideration AD pathogenesis, stages, symptoms, and risk factors.

The miR-181 family: Wide-ranging pathophysiological effects on cell fate and function.

MicroRNAs (miRNAs) are epigenetic regulators that can target and inhibit translation of multiple mRNAs within a given cell type. As such, a number of different pathways and networks may be modulated as a result. In fact, miRNAs are known to regulate many cellular processes including differentiation, proliferation, inflammation, and metabolism. This review focuses on the miR-181 family and provides information from the published literature on the role of miR-181 homologs in regulating a range of activities in different cell types and tissues. Of note, we have not included details on miR-181 expression and function in the context of cancer since this is a broad topic area requiring independent review. Instead, we have focused on describing the function and mechanism of miR-181 family members on differentiation toward a number of cell lineages in various non-neoplastic conditions (e.g., immune/hematopoietic cells, osteoblasts, osteoclasts, chondrocytes, adipocytes). We have also provided information on how modulation of miR-181 homologs can have positive effects on disease states such as cardiac abnormalities, pulmonary arterial hypertension, thrombosis, osteoarthritis, and vascular inflammation. In this context, we have used some examples of FDA-approved drugs that modulate miR-181 expression. We conclude by discussing some common mechanisms by which miR-181 homologs appear to regulate a number of different cellular processes and how targeting specific miR-181 family members may lead to attractive therapeutic approaches to treat a number of human disease or repair conditions, including those associated with the aging process.

Parallel synthesis of oligonucleotides containing N-acyl amino-LNA and their therapeutic effects as anti-microRNAs.

2'-Amino-locked nucleic acid (ALNA), maintains excellent duplex stability, and the nitrogen at the 2'-position is an attractive scaffold for functionalization. Herein, a facile and efficient method for the synthesis of various 2'-N-acyl amino-LNA derivatives by direct acylation of the 2'-amino moiety contained in the synthesized oligonucleotides and its fundamental properties are described. The introduction of the acylated amino-LNA enhances the potency of the molecules as therapeutic anti-microRNA oligonucleotides.

Macrophage miR-149-5p induction is a key driver and therapeutic target for BRONJ.

Bisphosphonate-related (BP-related) osteonecrosis of the jaw (BRONJ) is one of the severe side effects of administration of BPs, such as zoledronic acid (ZA), which can disrupt the patient's quality of life. Although the direct target of skeletal vasculature and bone resorption activity by BPs has been phenomenally observed, the underlying mechanism in BRONJ remains largely elusive. Thus, it is urgently necessary to discover effective therapeutic targets based on the multifaceted underlying mechanisms in the development of BRONJ. Here, we determined the inhibitory role of ZA-treated macrophages on osteoclast differentiation and type H vessel formation during tooth extraction socket (TES) healing. Mechanistically, ZA activated the NF-κB signaling pathway and then induced p65 nuclear translocation in macrophages to promote miR-149-5p transcription, resulting in impaired osteoclast differentiation via directly binding to the Traf6 3'-UTR region. Moreover, we identified that miR-149-5p-loaded extracellular vesicles derived from ZA-treated bone marrow-derived macrophages could regulate biological functions of endothelial cells via the Rap1a/Rap1b/VEGFR2 pathway. Furthermore, local administration of chemically modified antagomiR-149-5p was proven to be therapeutically effective in BRONJ mice. In conclusion, our findings illuminate the dual effects of miR-149-5p on skeletal angiogenesis and bone remolding, suggesting it as a promising preventive and therapeutic target for BRONJ.

A Tetrahedral Framework DNA-Based Bioswitchable miRNA Inhibitor Delivery System: Application to Skin Anti-Aging.

MicroRNA (miR)-based therapy shows strong potential; however, structural limitations pose a challenge in fully exploiting its biomedical functionality. Tetrahedral framework DNA (tFNA) has proven to be an ideal vehicle for miR therapy. Inspired by the ancient Chinese myth "Sun and Immortal Birds," a novel bioswitchable miR inhibitor delivery system (BiRDS) is designed with three miR inhibitors (the three immortal birds) and a nucleic acid core (the central sun). The BiRDS fuses miR inhibitors within the framework, maximizing their loading capacity, while allowing the system to retain the characteristics of small-sized tFNA and avoiding uncertainty associated with RNA exposure in traditional loading protocols. The RNase H-responsive sequence at the tail of each "immortal bird" enables the BiRDS to transform from a 3D to a 2D structure upon entering cells, promoting the delivery of miR inhibitors. To confirm the application potential, the BiRDS is used to deliver the miR-31 inhibitor, with antiaging effects on hair follicle stem cells, into a skin aging model. Superior skin penetration ability and RNA delivery are observed with significant anti-aging effects. These findings demonstrate the capability and editability of the BiRDS to improve the stability and delivery efficacy of miRs for future innovations.

Inhibition of miR-let-7i Induces DC Immature Cells and Improves Skin Graft Tolerance.

Dendritic cells (DC) initiate the immune response in the body. They can stimulate T cell activation, proliferation, and differentiation and ultimately participate in the immune response and the immune tolerance response. The purpose of this study was to coculture DCs and T cells and subcutaneously inject DCs transfected with miR-let-7i into rhesus monkey transplantations to verify the role of miR-let-7i in allograft immune tolerance. In vitro studies found that the expression of miR-let-7i was upregulated after inducing the maturation of DCs. The low expression of miR-let-7i inhibited the maturation of DCs, promoted the differentiation of T cells into T helper T cells 2 (Th2), and inhibited T helper T cell 1- (Th1-) driven rejection. In vivo studies also obtained similar results, and subcutaneous injection of DCs transfected with miR-let-7i inhibitor prolonged the survival time of allogeneic skin transplantation. Therefore, we conclude that inhibition of miR-let-7i inhibits DC maturation and improves the tolerance of grafted skin.

Identification of benzamides derivatives of norfloxacin as promising microRNA-21 inhibitors via repressing its transcription.

MicroRNA-21 is a carcinogenic microRNA, whose overexpression arises in a variety of tumor tissues. Hence, microRNA-21 a prospective target for cancer treatment, and regulation of microRNA-21 by small molecule inhibitors is deemed as a promising approach for tumor therapy. In this work, to discover potent microRNA-21 inhibitor, series of 4-(N-norfloxacin-acyl)aminobenzamides were designed and synthesized, and their inhibitory effects were appraised by utilizing dual luciferase reporter assays. The results indicated that compound A7 was the most efficient microRNA-21 small molecule inhibitor. What's more, A7 suppressed the migration of Hela cells and the colony formation of Hela and HCT-116 cells as well as promoted apoptosis of Hela cells. In the mechanism study, results of RT-qPCR certified that A7 could reduce the level of mature microRNA-21 via disrupting its expression at the transcriptional level of its primary form "pri-miR-21", which was distinct from most previous inhibitors directly binding with pre-miR-21. Noticeably, Western blotting and RT-qPCR uncovered A7 could upregulate the expression PTEN, EGR1 and SLIT2, which are the downstream functional targets of microRNA-21. These findings demonstrated that A7 was a promising microRNA-21 small molecule inhibitor and 4-(N-norfloxacin-acyl) aminobenzamide can serve as a new scaffold for discovery of potent microRNA-21 inhibitor.

Exosomal miR-17-3p Alleviates Programmed Necrosis in Cardiac Ischemia/Reperfusion Injury by Regulating TIMP3 Expression.

OBJECTIVE: Myocardial ischemia/reperfusion (I/R) injury can aggravate myocardial injury. Programmed necrosis plays a crucial role in this injury. However, the role of exosomal miRNAs in myocardial I/R injury remains unclear. Therefore, this study is aimed at exploring the function and mechanism of exosomal miR-17-3p in myocardial I/R injury. METHODS: The myocardial I/R injury animal model was established in C57BL/6 mice. Exosomes were identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Programmed necrosis was detected by PI staining. Heart function and myocardial infarct size were evaluated using echocardiography and triphenyl tetrazolium chloride (TTC) staining, respectively. Histopathological changes were visualized by hematoxylin and eosin (H&E) and Masson staining. The regulation of TIMP3 expression by miR-17-3p was verified using a dual-luciferase reporter assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assays (ELISA). TIMP3 expression was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. RESULTS: We demonstrated that miR-17-3p was significantly downregulated in peripheral blood exosomes after cardiac I/R injury. Further analysis indicated that exosomal miR-17-3p attenuated H2O2-induced programmed necrosis in cardiomyocytes in vitro. Moreover, TIMP3 was a target for miR-17-3p. TIMP3 affected H2O2-induced programmed necrosis in cardiomyocytes. This effect was modulated by miR-17-3p in vitro. Furthermore, exosomal miR-17-3p greatly alleviated cardiac I/R injury in vivo. CONCLUSIONS: The present study demonstrated that exosomal miR-17-3p alleviated the programmed necrosis associated with cardiac I/R injury by regulating TIMP3 expression. These findings could represent a potential treatment for I/R injury.

Administration of a microRNA-21 inhibitor improves the lupus-like phenotype in MRL/lpr mice by repressing Tfh cell-mediated autoimmune responses.

BACKGROUND: Inhibiting Tfh cell overexpansion prevents autoimmune responses and disease flares in systemic lupus erythematosus (SLE). miR-21 is highly expressed in SLE CD4+ T cells, but whether inhibiting miR-21 can reduce Tfh cell expansion and alleviate the disease progression of lupus is unclear. AIM OF THE STUDY: To address the role and molecular mechanism of miR-21 in regulating Tfh cell expansion and its therapeutic effect on SLE. METHODS: We treated 12-week-old MRL/lpr mice with Antagomir-21, which specifically inhibited miR-21 in vivo. After 12 weeks of treatment, we examined the proportions of Tfh cells and germinal center (GC) B cells and serum levels of autoantibodies and evaluated disease severity by histological scoring and albuminuria. We determined the level of intracellular free iron in CD4+ T cells by PGSK probe and examined the expression of the Fth and Tfrc genes by qPCR. Immunohistochemistry (IHC)was used to assess the 5-hmC level in the draining lymph nodes (dLNs) and spleen. RESULTS AND CONCLUSIONS: Inhibiting miR-21 significantly reduced the expansion of Tfh cells and GC B cells. Furthermore, Antagomir-21 highly improved skin lesions and nephritis in MRL/lpr mice. Inhibiting miR-21 reduced intracellular iron accumulation and DNA hydroxymethylation in T cells. In conclusion, inhibiting miR-21 in vivo improves intracellular iron homeostasis and inhibits Tfh cell overexpansion, contributing to reduced autoimmune responses and the remission of disease symptoms in murine lupus.

Inhibition of miRNA-100 facilitates bone regeneration defects of mesenchymal stem cells in osteoporotic mice through the protein kinase B pathway.

Osteoporotic patients suffer from bone microstructure damage and are prone to fracture and bone defect. Due to the damage of bone healing ability, the bone repair of osteoporotic patients is usually slow. Here we aimed to explore the function and potential molecular mechanism of miR-100 in osteogenic differentiation ability of bone marrow stem cells (BMSCs). Ovariectomy was performed on mice to induce osteoporosis. BMSCs were extracted from normal and ovariectomized (OVX) mice to examine the effect of microRNA (miR)-100 on BMSC osteogenic differentiation. Hematoxylin and eosin (H&E) staining and safranin O-fast green staining assays were performed on femur tissues to reveal pathological changes. The osteogenic differentiation of BMSCs were determined by Alkaline Phosphatase and Alizarin red staining assays. The results showed that miR-100 expression was significantly upregulated in bone tissues and BMSCs from osteoporotic mice. MiR-100 knockdown partially improved osteogenic function of OVX mice-derived BMSCs. Next, mechanistic target of rapamycin kinase (MTOR) was identified as the target downstream miR-100. MiR-100 deficiency can activate the protein kinase B (AKT)/mTOR pathway. MiR-100 controlled the osteogenic function of BMSCs by the AKT/mTOR pathway. Collectively, our findings demonstrate that inhibition of miR-100 facilitates bone regeneration defects of BMSCs in osteoporotic mice through AKT pathway, indicating that miR-100 might be an effective target for the treatment of osteoporotic mandibular injury and bone defect diseases.

SOCS6 Promotes Mitochondrial Fission and Cardiomyocyte Apoptosis and Is Negatively Regulated by Quaking-Mediated miR-19b.

BACKGROUND: Mitochondrial dysfunction and abnormal mitochondrial fission have been implicated in the complications associated with I/R injury as cardiomyocytes are abundant in mitochondria. SOCS6 is known to participate in mitochondrial fragmentation, but its exact involvement and the pathways associated are uncertain. METHODS AND RESULTS: The expression of SOCS6 was analyzed by western blot in cardiomyocytes under a hypoxia and reoxygenation (H/R) model. A dual-luciferase reporter assay was used to confirm the direct interaction between miR-19b and the 3'-UTR of Socs6. In the present study, we found that Socs6 inhibition by RNA interference attenuated H/R-induced mitochondrial fission and apoptosis in cardiomyocytes. A luciferase assay indicated that Socs6 is a direct target of miR-19b. The overexpression of miR-19b decreased mitochondrial fission and apoptosis in vitro. Moreover, the presence of miR-19b reduced the level of SOCS6 and the injury caused by I/R in vivo. There were less apoptotic cells in the myocardium of mice injected with miR-19b. In addition, we found that the RNA-binding protein, Quaking (QK), participates in the regulation of miR-19b expression. CONCLUSIONS: Our results indicate that the inhibition of mitochondrial fission through downregulating Socs6 via the QK/miR-19b/Socs6 pathway attenuated the damage sustained by I/R. The QK/miR-19b/Socs6 axis plays a vital role in regulation of mitochondrial fission and cardiomyocyte apoptosis and could form the basis of future research in the development of therapies for the management of cardiac diseases.

Cardioprotective Effect of circ_SMG6 Knockdown against Myocardial Ischemia/Reperfusion Injury Correlates with miR-138-5p-Mediated EGR1/TLR4/TRIF Inactivation.

Increased neutrophil recruitment represents a hallmark event in myocardial ischemia/reperfusion (I/R) injury due to the ensuing inflammatory response. Circular RNAs (circRNAs) are important regulatory molecules involved in cell physiology and pathology. Herein, we analyzed the role of a novel circRNA circ_SMG6 in the regulation of neutrophil recruitment following I/R injury, which may associate with the miR-138-5p/EGR1/TLR4/TRIF axis. Myocardial I/R injury was modeled in vivo by ligation of the left anterior descending (LAD) artery followed by reperfusion in mice and in vitro by exposing a cardiomyocyte cell line (HL-1) to hypoxia/reoxygenation (H/R). Gain- and loss-of-function experiments were performed to evaluate the effect of the circ_SMG6/miR-138-5p/EGR1/TLR4/TRIF axis on cardiac functions, myocardial infarction, myocardial enzyme levels, cardiomyocyte activities, and neutrophil recruitment. We found that the EGR1 expression was increased in myocardial tissues of I/R mice. Knockdown of EGR1 was found to attenuate I/R-induced cardiac dysfunction and infarction area, pathological damage, and cardiomyocyte apoptosis. Mechanistic investigations showed that circ_SMG6 competitively bound to miR-138-5p and consequently led to upregulation of EGR1, thus facilitating myocardial I/R injury in mice and H/R-induced cell injury. Additionally, ectopic EGR1 expression augmented neutrophil recruitment and exacerbated the ensuing I/R injury, which was related to the activated TLR4/TRIF signaling pathway. Overall, our findings suggest that circ_SMG6 may deteriorate myocardial I/R injury by promoting neutrophil recruitment via the miR-138-5p/EGR1/TLR4/TRIF signaling. This pathway may represent a potential therapeutic target in the management of myocardial I/R injury.

miR-340-5p Mediates Cardiomyocyte Oxidative Stress in Diabetes-Induced Cardiac Dysfunction by Targeting Mcl-1.

Diabetic cardiomyopathy (DCM) is initially characterized by early diastolic dysfunction, left ventricular remodeling, hypertrophy, and myocardial fibrosis, and it is eventually characterized by clinical heart failure. MicroRNAs (miRNAs), endogenous small noncoding RNAs, play significant roles in diabetes mellitus (DM). However, it is still largely unknown about the mechanism that links miRNAs and the development of DCM. Here, we aimed to elucidate the mechanism underlying the potential role of microRNA-340-5p in DCM in db/db mouse, which is a commonly used model of type 2 DM and diabetic complications that lead to heart failure. We first demonstrated that miR-340-5p expression was dramatically increased in heart tissues of mice and cardiomyocytes under diabetic conditions. Overexpression of miR-340-5p exacerbated DCM, which was reflected by extensive myocardial fibrosis and more serious dysfunction in db/db mice as represented by increased apoptotic cardiomyocytes, elevated ROS production, and impaired mitochondrial function. Inhibition of miR-340-5p by a tough decoy (TUD) vector was beneficial for preventing ROS production and apoptosis, thus rescuing diabetic cardiomyopathy. We identified myeloid cell leukemia 1 (Mcl-1) as a major target gene for miR-340-5p and showed that the inhibition of Mcl-1 was responsible for increased functional loss of mitochondria, oxidative stress, and cardiomyocyte apoptosis, thereby caused cardiac dysfunction in diabetic mice. In conclusion, our results showed that miR-340-5p plays a crucial role in the development of DCM and can be targeted for therapeutic intervention.

miR-154-5p Affects the TGFß1/Smad3 Pathway on the Fibrosis of Diabetic Kidney Disease via Binding E3 Ubiquitin Ligase Smurf1.

AIM: The study is aimed at verifying miR-154-5p and Smurf1 combination in glomerular mesangial cells regulating TGFß1/Smad3 pathway-related protein ubiquitination in the model of diabetic rats renal tissues, primary mesangial cells, and cell lines. METHODS: The diabetic SD rat model and high-glucose-cultured primary mesangial cells and cell lines were established. miR-154-5p mimic and inhibitor, Smurf1 siRNA, and TGF ß 1/Smad3 inhibitor (SB431542) were pretreated to make the TGFß1/Smad3 pathway and ubiquitin changes. Fluorescence in situ hybridization was used for the miR-154-5p renal localization; molecular biological detection was adopted for cell proliferation, renal function, urine protein, and pathway proteins. After bioinformatics predicted binding sites, luciferase and Co-IP were used to detect miRNA and protein binding. RESULTS: miR-154-5p was significantly increased and mainly concentrated in the glomerular of renal cortex in well-established diabetic rat renal tissues. Rno-miR-154-5p combined Rno-Smurf1 3' UTR, while Smurf1 combined Smad3 directly. Meanwhile, miR-154-5p regulates TGFß1/Smad3-mediated cell proliferation via Smurf1 ubiquitination. CONCLUSION: miR-154-5p regulates the TGFß1/Smads pathway through Smurf1 ubiquitination and promotes the fibrosis process of diabetic kidney disease.

Exosomal MALAT1 Derived from High Glucose-Treated Macrophages Up-Regulates Resistin Expression via miR-150-5p Downregulation.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a crucial role in the pathophysiological process associated with diabetes-related complications. The effect of high glucose levels on macrophage-derived exosomal MALAT1 is unknown. Therefore, we investigated the molecular regulatory mechanisms controlling exosomal MALAT1 in macrophages under high glucose treatment and the therapeutic target of macrophage-derived exosomal MALAT1 using a balloon injury model of vascular disease in diabetic rats. High glucose (25 mM) significantly increased MALAT1 expression in macrophage-derived exosomes. MALAT1 suppressed miR-150-5p expression in macrophage-derived exosomes under high-glucose conditions. Silencing MALAT1 using MALAT1 siRNA significantly reversed miR-150-5p expression induced by macrophage-derived exosomes. Macrophage-derived exosomes under high-glucose treatment significantly increased resistin expression in macrophages. Silencing MALAT1 and overexpression of miR-150-5p significantly decreased resistin expression induced by macrophage-derived exosomes. Overexpression of miR-150-5p significantly decreased resistin luciferase activity induced by macrophage-derived exosomes. Macrophage-derived exosome significantly decreased glucose uptake in macrophages and silencing MALAT1, resistin or overexpression of miR-150-5p significantly reversed glucose uptake. Balloon injury to the carotid artery significantly increased MALAT1 and resistin expression and significantly decreased miR-150-5p expression in arterial tissue. Silencing MALAT1 significantly reversed miR-150-5p expression in arterial tissue after balloon injury. Silencing MALAT1 or overexpression of miR-150-5p significantly reduced resistin expression after balloon injury. In conclusion, high glucose up-regulates MALAT1 to suppress miR-150-5p expression and counteracts the inhibitory effect of miR-150-5p on resistin expression in macrophages to promote vascular disease. Macrophage-derived exosomes containing MALAT1 may serve as a novel cell-free approach for the treatment of vascular disease in diabetes mellitus.

γ Peptide Nucleic Acid-Based miR-122 Inhibition Rescues Vascular Endothelial Dysfunction in Mice Fed a High-Fat Diet.

The blood levels of microRNA-122 (miR-122) is associated with the severity of cardiovascular disorders, and targeting it with efficient and safer miR inhibitors could be a promising approach. Here, we report the generation of a γ-peptide nucleic acid (γPNA)-based miR-122 inhibitor (γP-122-I) that rescues vascular endothelial dysfunction in mice fed a high-fat diet. We synthesized diethylene glycol-containing γP-122-I and found that its systemic administration counteracted high-fat diet (HFD)-feeding-associated increase in blood and aortic miR-122 levels, impaired endothelial function, and reduced glycemic control. A comprehensive safety analysis established that γP-122-I affects neither the complete blood count nor biochemical tests of liver and kidney functions during acute exposure. In addition, long-term exposure to γP-122-I did not change the overall adiposity, or histology of the kidney, liver, and heart. Thus, γP-122-I rescues endothelial dysfunction without any evidence of toxicity in vivo and demonstrates the suitability of γPNA technology in generating efficient and safer miR inhibitors.