Increased Expression Of miR-126 Research Articles

Up-regulation of miR-126 via DNA methylation in hypoxia-preconditioned endothelial cells may contribute to hypoxic tolerance of neuronal cells.

Endothelial cells (ECs) can confer neuroprotection by secreting molecules. This study aimed to investigate whether DNA methylation contributes to the neuroprotective gene expression induced by hypoxia preconditioning (HPC) in ECs and to clarify that the secretion of molecules from HPC ECs may be one of the molecular mechanisms of neuroprotection. Human microvascular endothelial cell-1 (HMEC-1) was cultured under normal conditions (C), hypoxia(H), and hypoxia preconditioning (HPC), followed by the isolation of culture medium (CM). SY5Y cell incubated with the isolated CM from HMEC-1 was exposed to oxygen-glucose deprivation (OGD). The DNA methyltransferases (DNMTs), global methylation level, miR-126 and its promotor DNA methylation level in HMEC-1 were measured. The cell viability and cell injury in SY5Y were detected. HPC decreased DNMTs level and global methylation level as well as increased miR-126 expression in HMEC-1. CM from HPC treated HMEC-1 also relieved SY5Y cell damage, while CM from HMEC-1 which over-expression of miR-126 can reduce injury in SY5Y under OGD condition. These findings indicate EC may secrete molecules, such as miR-126, to execute neuroprotection induced by HPC through regulating the expression of DNMTs.

Effects of miR126 Expressing Adipose-Derived Stem Cells on Fat Graft Survival and Angiogenesis.

Fat transplantation supported by supplementation with ASCs has become a reliable procedure for treating soft tissue defects. However, the unpredictable survival rates for grafted fat remains a challenge with post-transplantation ischemia causing tissue loss. MiR126, which regulates VEGF signaling, is an endothelial cell-specific miRNA known to play an essential role in angiogenesis. We hypothesized that increased miR126 expression in grafted ASCs may promote fat survival within an autologous fat transfer model. Rat adipose-derived stem cells were isolated, expanded ex vivo for three passages and then transduced with miR126. We used PCR to verify lentiviral transduction and ELISA to confirm VEGF expression. We then mixed autologous fat tissues from our rat model with transduced ASCs, augmented with a nonsense control or miR126 expression vector. These mixtures were used in the fat grafting procedure, completed via subcutaneous injection at three paravertebral points in each rat. Fat grafts were then harvested on days 4, 7, 14, and 28 post-transplant and evaluated for survival, neovascularization, and protein expression via western blot. VEGF expression levels in ASCs, Con-ASCs, and miR126-ASCs were not significantly different. However, miR126-ASCs experienced significantly improved survival on days 7, 14, and 28 when compared with the other groups. These ASCs also presented with the greatest capillary density on days 7, 14, and 28 post-transplantation as well as increased p-ERK and p-AKT expression when compared to the other groups. This data suggests that miR126 augmentation of ASCs may help to enhance the survival and angiogenic capacity of transplanted fat tissues, and that this augmentation was not dependent on VEGF but rather the activation of the ERK/AKT pathway. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The effects of all trans retinoic acid, vitamin D3 and their combination on plasma levels of miRNA-125a-5p, miRNA-34a, and miRNA-126 in an experimental model of diabetes.

Objective:The purpose of this study was to evaluate the effects of ATRA (all trans retinoic acid), vitamin D3, and their combination on circulating levels of miR (MicroRNA) -125a-5p, miR-126, and miR-34ain diabetic rats.Materials and Methods:Total miRNA was extracted from plasma samples. miRNA expression profiles of 30 rats in five groups were analyzed after 4-week intervention. The expression levels of miRNAs were measured using qRT-PCR.Results:We analyzed the expression of miR-126, miR-125a-5p, and miR-34a in serum among all five groups (p=0.268). The levels of miRNA-126 (p=0.004) and miR-125a-5p (p=0.014) showed a significant difference among our experimental groups. The circulating levels of miR-126 decreased in DC (Diabetic control) group compared to the HC (Healthy control) group (p=0.009). In addition, vitamin D3+ATRA supplementation increased miR-126 expression (p=0.014). Moreover, the levels of miR-125a-5p decreased in the DC group compared to the HC group (p=0.019). Conclusion:The expression of miR-126 and miR-125a-5p decreased in diabetic rats. Also, vitamin D3+ATRA can be considered a new therapeutic agent that can elevate miR-126 expression and prevent diabetes-related cardiovascular complications.

MiR-126 contributes to the epigenetic signature of diabetic vascular smooth muscle and enhances antirestenosis effects of Kv1.3 blockers

ObjectivesRestenosis after vessel angioplasty due to dedifferentiation of the vascular smooth muscle cells (VSMCs) limits the success of surgical treatment of vascular occlusions. Type 2 diabetes (T2DM) has a major impact on restenosis, with patients exhibiting more aggressive forms of vascular disease and poorer outcomes after surgery. Kv1.3 channels are critical players in VSMC proliferation. Kv1.3 blockers inhibit VSMCs MEK/ERK signalling and prevent vessel restenosis. We hypothesize that dysregulation of microRNAs (miR) play critical roles in adverse remodelling, contributing to Kv1.3 blockers efficacy in T2DM VSMCs. Methods and resultsWe used clinically relevant in vivo models of vascular risk factors (VRF) and vessels and VSMCs from T2DM patients. ResuktsHuman T2DM vessels showed increased remodelling, and changes persisted in culture, with augmented VSMCs migration and proliferation. Moreover, there were downregulation of PI3K/AKT/mTOR and upregulation of MEK/ERK pathways, with increased miR-126 expression. The inhibitory effects of Kv1.3 blockers on remodelling were significantly enhanced in T2DM VSMCs and in VRF model. Finally, miR-126 overexpression confered “diabetic” phenotype to non-T2DM VSMCs by downregulating PI3K/AKT axis. ConclusionsmiR-126 plays crucial roles in T2DM VSMC metabolic memory through activation of MEK/ERK pathway, enhancing the efficacy of Kv1.3 blockers in the prevention of restenosis in T2DM patients.

Long non-coding RNA HOTAIR promotes the progression of synovial sarcoma through microRNA-126/stromal cell-derived factor-1 regulation.

The long non-coding RNA (lncRNA) HOTAIR is an oncogene, that has been reported to be aberrantly expressed in multiple types of malignant tumor tissues. However, its expression and association with synovial sarcoma (SS) remains unclear. The present study aimed to elucidate the expression level of HOTAIR in SS tissues and also identify its role. Reverse transcription-quantitative PCR was used to detect the expression level of HOTAIR and microRNA (miR)-126 in 54 tissue samples from patients with SS, in 10 tissue samples from synovium tissues of normal patients, and in SW982 cells. The protein expression level was measured using western blot analysis and cellular immunofluorescence. Cellular proliferation, invasion and migration were assessed using MTT, Transwell and wound healing assays, respectively. HOTAIR was expressed at high levels in SS tissues. In contrast, miR-126 was expressed at low levels in SS tissues, and was negatively correlated with HOTAIR expression. HOTAIR knockdown in SW982 cells inhibited cellular proliferation in vitro, but also significantly increased the ratio of cells in the G1/G0 phase of the cell cycle, and decreased the ratio of cells in the G2/S phase. In addition, HOTAIR knockdown inhibited the invasion and migration of the SW982 cells, as observed in the Transwell and wound healing assays. Furthermore, HOTAIR knockdown increased miR-126 expression level and decreased the expression level of stromal cell-derived factor-1 (SDF-1) at the protein level. On the other hand, while miR-126-mimic decreased the protein expression level of SDF-1, miR-126-inhibitor increased its expression level in SW982 cells. Notably, HOTAIR knockdown or SDF-1 knockout significantly decreased the protein expression levels of CDK1, CDK2, cyclin D1, MMP-9, vimentin and N-cadherin, and significantly increased the protein expression levels of p21, p53 and E-cadherin in SW982 cells. HOTAIR was highly expressed in SS tissues, wherein it could promote the proliferation, invasion and migration of SS cells by increasing the expression of SDF-1 via miR-126 inhibition.

Aerobic exercise leads to upregulation of Mir-126 and angiogenic signaling in the heart tissue of diabetic rats

ObjectiveMicroRNA-126 plays a critical role in promoting cardiac angiogenesis. Aerobic exercise has also cardioprotective effects by upregulating angiogenic factors. The present study aimed to evaluate the impact of aerobic exercise on cardiac expression of mir-126 and angiogenic signaling pathways. MethodsThirty-two male Wistar rats (180–240 g) were randomly divided into four diabetic control, healthy control, healthy exercise, and diabetic exercise groups. Diabetes was induced by intraperitoneal injection of Streptozotocin (STZ) 50 mg/kg (with citrate buffer) and nicotinamide 110 mg/kg (with saline). Aerobic training groups were performing on a rodent treadmill with an intensity of 25 m/min, 5° gradients, and 30 min each session for 8 weeks. The control groups were placed on a stationary treadmill without training. The heart of the rats was taken and the expression level of cardiac mir-126 was evaluated by Real-time PCR. The protein levels of raf1, spred1, and VEGF were measured using ELISA. Immunohistochemistry was used to measure cardiac muscle capillary density. ResultsThe results showed that diabetes induced by STZ disrupted angiogenesis signaling through the reduction of mir-126, raf1, and VEGF, while an increase in spred1 protein level (p = 0.01 for all). However, 8 weeks of aerobic exercise (in healthy animals) significantly increased mir-126 expression, and raf1 and VEGF proteins (p = 0.05), while significantly decreased spred1 protein (p = 0.05). Also, 8 weeks of exercise training in diabetic rats might improve the cardiac tissue by increasing mir-126 and angiogenic signaling pathways. ConclusionThe results of this study suggest that aerobic exercise training may prevent negative effects of diabetes on heart tissue by improving angiogenic pathway. Therefore, aerobic exercise may be a good strategy for coping with cardiovascular cell damage of diabetes.

MiR-126/VCAM-1 regulation by naringin suppresses cell growth of human non-small cell lung cancer.

Certain studies have indicated that naringin possesses various pharmacological activities including anti-aging, anti-oxidation, anticancer, cardiovascular and cerebrovascular disease prevention, in addition to anti-hepatic effects. The present study explores the anticancer effect of naringin on human small cell lung cancer H69AR cells. Cell growth and apoptosis rates of H69AR cells were measured by MTT or flow cytometry, which demonstrated naringin suppressed cell growth and induced apoptosis of H69AR cells. MicroRNA (miR)-126 expression and levels of phosphorylated protein kinase B (AKT), mechanistic target of rapamycin (mTOR), nuclear factor (NF)-κB and vascular cell adhesion molecule 1 (VCAM-1) proteins were detected by quantitative polymerase chain reaction and western blotting. It was identified that naringin increased miR-126 expression and suppressed the phosphorylation of AKT, mTOR, NF-κB and VCAM-1 proteins in H69AR cells. Suppression of miR-126 expression reduced the anticancer effects of naringin on H69AR cells, reversed the naringin-induced reduction of phosphoinositide 3-kinase/AKT/mTOR, and suppressed VCAM-1 protein levels. However, close of miR-126 expression did not affect the levels of NF-κB protein in H69AR cells. In summary, naringin exhibits its anti-cancer effect by suppressing cell growth of small cell lung cancer cells through miR-126/VCAM-1 signaling pathway.

A post-weaning obesogenic diet exacerbates the detrimental effects of maternal obesity on offspring insulin signaling in adipose tissue

Previous studies have shown that maternal diet-induced obesity leads to increased risk of type 2 diabetes in offspring. The current study investigated if weaning onto an obesogenic diet exaggerated the detrimental effects of maternal diet-induced obesity in adipose tissue. Maternal obesity and offspring obesity led to reduced expression of key insulin signalling proteins, including insulin receptor substrate-1 (IRS-1). The effects of maternal obesity and offspring obesity were, generally, independent and additive. Irs1 mRNA levels were similar between all four groups of offspring, suggesting that in both cases post-transcriptional regulation was involved. Maternal diet-induced obesity increased miR-126 expression however levels of this miR were not influenced by a post-weaning obesogenic diet. In contrast, a post-weaning obesogenic diet was associated with increased levels of suppressor of cytokine signaling-1, implicating increased degradation of IRS-1 as an underlying mechanism. Our results suggest that whilst programmed reductions in IRS-1 are associated with increased levels of miR-126 and consequently reduced translation of Irs1 mRNA, the effects of a post-weaning obesogenic diet on IRS-1 are mediated by miR-126 independent mechanisms, including increased IRS-1 protein degradation. These divergent mechanisms explain why the combination of maternal obesity and offspring obesity leads to the most pronounced effects on offspring metabolism.

Increasing the miR-126 expression in the peripheral blood of patients with diabetic foot ulcers treated with maggot debridement therapy

BackgroundmiR-126 may increase angiogenesis in patients with diabetic foot ulcers (DFUs) treated with maggot debridement therapy (MDT). MethodsReal-time quantitative PCR was used to detect expression of miR-126 mRNA in the peripheral blood among the non-diabetic population, type 2 diabetes mellitus patients without DFU, and patients with DFUs of type 2 diabetes mellitus. The expression of miR-126 mRNA in the peripheral blood of patients with DFUs was observed before and after MDT. Finally, human umbilical vein endothelial cells (HUVEC) were utilized to explore miR-126 mRNA expression with maggot excretions/secretions (ES). ResultsIn the patients with DFUs, the miR-126 mRNA expression level in the peripheral blood was less than that type 2 diabetes mellitus patients without DFU, and much lower than that in the non-diabetic population (P<0.001). The miR-126 expression level was significantly increased in those DFU patients treated with MDT (P<0.05). Finally, using HUVEC co-cultured with ES, we showed the ES increased miR-126 expression in vitro (P<0.001). ConclusionMDT upregulates the miR-126 expression in the peripheral blood of patients with DFUs.

A Stromal Cell-Derived Factor 1α Analogue Improves Endothelial Cell Function in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome.

Endothelial cell (EC) dysfunction is a critical mediator of the acute respiratory distress syndrome (ARDS). Recent studies have demonstrated that stromal cell-derived factor 1α (SDF-1α) promotes EC barrier integrity. Our previous studies used a SDF-1α analogue CTCE-0214 (CTCE) in experimental sepsis and demonstrated that it attenuated vascular leak and modulated microRNA (miR) levels. We examined the hypothesis that CTCE improves EC function in lipopolysaccharide (LPS)-induced ARDS through increasing miR-126 expression. Human microvascular endothelial cells (HMVECs) were treated with thrombin to disrupt the EC integrity followed by incubation with CTCE or SDF-1α. Barrier function was determined by trans-endothelial electrical resistance assay. CTCE-induced alterations in miRNA expression and signaling pathways involved in barrier function were determined. Thrombin-induced vascular leak was abrogated by both CTCE and SDF-1α. CTCE also prevented thrombin-induced decreases of vascular endothelial (VE)-cadherin cell surface expression and expansion of the intercellular space. CTCE increased miR-126 levels and induced activation of AKT/Rac 1 signaling. Cotreatment with a miR-126 inhibitor blocked the protective effects of CTCE on AKT activation and endothelial permeability. In subsequent in vivo studies, ARDS was induced by intratracheal instillation of LPS. Intravenous injection of CTCE diminished the injury severity as evidenced by significant reductions in protein, immune cells, inflammatory cytokines and chemokines in the bronchoalveolar lavage fluid, increased miR-126 expression and decreased pulmonary vascular leak and alveolar edema. Taken together, our data show that CTCE improves endothelial barrier integrity through increased expression of miR-126 and activation of Rac 1 signaling and represents an important potential therapeutic strategy in ARDS.

MiR-126 Contributes to Human Umbilical Cord Blood Cell-Induced Neurorestorative Effects After Stroke in Type-2 Diabetic Mice.

Diabetes mellitus (DM) is a high risk factor for stroke and leads to more severe vascular and white-matter injury than stroke in non-DM. We tested the neurorestorative effects of delayed human umbilical cord blood cell (HUCBC) treatment of stroke in type-2 diabetes (T2DM). db/db-T2DM and db/+-non-DM mice were subjected to distal middle cerebral artery occlusion (dMCAo) and were treated 3 days after dMCAo with: (a) non-DM + Phosphate buffered saline (PBS); (b) T2DM + PBS; (c) T2DM + naïve-HUCBC; (d) T2DM + miR-126(-/-) HUCBC. Functional evaluation, vascular and white-matter changes, neuroinflammation, and miR-126 effects were measured in vivo and in vitro. T2DM mice exhibited significantly decreased serum and brain tissue miR-126 expression compared with non-DM mice. T2DM + HUCBC mice exhibited increased miR-126 expression, increased tight junction protein expression, axon/myelin, vascular density, and M2-macrophage polarization. However, decreased blood-brain barrier leakage, brain hemorrhage, and miR-126 targeted gene vascular cell adhesion molecule-1 and monocyte chemotactic protein 1 expression in the ischemic brain as well as improved functional outcome were present in HUCBC-treated T2DM mice compared with control T2DM mice. MiR-126(-/-) HUCBC-treatment abolished the benefits of naïve-HUCBC-treatment in T2DM stroke mice. In vitro, knock-in of miR-126 in primary cultured brain endothelial cells (BECs) or treatment of BECs with naïve-HUCBCs significantly increased capillary-like tube formation, and increased axonal outgrowth in primary cultured cortical neurons; whereas treatment of BECs or cortical neurons with miR-126(-/-) HUCBC attenuated HUCBC-treatment-induced capillary tube formation and axonal outgrowth. Our data suggest delayed HUCBC-treatment of stroke increases vascular/white-matter remodeling and anti-inflammatory effects; MiR-126 may contribute to HUCBC-induced neurorestorative effects in T2DM mice.

Abstract 165: Microrna-126 Contributes to Human Umbilicus Cord Blood Cell Induced Neurorestorative Effects in Type Two Diabetes Mice

Introduction: Human umbilical cord blood cell (HUCBC) treatment of stroke induces neurorestorative effects in non-diabetic rats. Whether HUCBC induces neurorestorative effects in type two diabetes (T2DM) has not been investigated. In this study, we tested the neurorestorative therapeutic effects and underlying mechanisms of action of HUCBC treatment of stroke in T2DM mice. Methods: BKS.Cg-m+/+Lepr db /J (db/db)-T2DM mice were subjected to extraluminal permanent distal MCAo ( dMCAo ) and were treated with: 1) PBS control; 2) HUCBC (1x10 6 ) at 3 days after dMCAo. A battery of functional tests was performed. Mice were sacrificed at 14 days after dMCAo. Vascular and white matter (WM) changes and microRNA were measured. Results: HUCBC treatment of stroke in T2DM mice does not decrease lesion volume (12.7±3.0% vs control: 14.5±3.2%), but significantly improves functional outcome, increases axon (Bielschowsky silver), myelin (Luxol fast blue) and arteriolar (a-SMA) density in the ischemic boundary zone (IBZ) compared to T2DM-dMCAo control mice (p&lt;0.05). To test the mechanisms of HUCBC induced neurorestorative effects, serum and ischemic brain miR-126 expression were measured. T2DM mice exhibit significantly decreased miR-126 expression in serum and in the IBZ compared to db+ (no-DM) control mice (p&lt;0.05). While HUCBC treatment in T2DM mice significantly increased miR-126 expression compared to non-treatment T2DM mice. In vitro, we found that HUCBC secretes miR-126 enriched exosomes and also stimulates brain endothelial cell miR-126 expression. Co-culture of brain endothelial cells (BECs) with primary cortical neurons (PCNs) increases PCN axon outgrowth. Knockdown of miR-126 in BECs not only decreases capillary tube formation, but also decreases PCN axonal outgrowth compared to miR-126-/-Con-BECs (p&lt;0.05). Knockdown miR-126 in HUCBC (miR-126-/-HUCBC) treatment of stroke in T2DM mice significantly decreases miR-126 expression in blood serum and attenuates HUCBC induced functional outcome after stroke in T2DM mice. Conclusion: HUCBC treatment initiated 3 days after stroke improves functional outcome, increases WM and vascular remodeling in T2DM mice. Increasing miR-126 contribute to HUCBC induced neurorestorative effects in T2DM.

Abstract 359: Human CD34+ Cell-Derived Exosomes Target Endothelial Cells to Deliver MiR-126 Promoting Revascularization and Myocardial Repair

Introduction: Locally transplanted human CD34 + stem cells have been shown to improve exercise tolerance in patients with myocardial ischemia and promote angiogenesis in animal models. Recently we have demonstrated that CD34 + cells secrete exosomes, membrane bound nano-vesicles, as a major component of their paracrine secretion which induces angiogenesis. We hypothesize that cell-free exosomes from CD34 + cells (CD34 Exo) mimic the beneficial effects of cells, and promote myocardial revascularization and repair via transfer of pro-angiogenic microRNAs, possibly to endothelial cells. Methods and Results: Therapeutic potential of CD34 Exo isolated from equal number of adult human peripheral blood-derived CD34 + cells was evaluated in a murine model of myocardial ischemia (MI). Similar to cells, treatment with CD34 Exo resulted in significant improvement in ischemia compared to treatment with PBS (ejection fraction, 42±4 v 22±6%; capillary density, 113±7 v 66±6/HPF; fibrosis 27±2 v 48±7%, p&lt;0.05, n=7-12). Interestingly, confocal imaging and flow cytometry analyses revealed that CD34 Exo was selectively internalized by endothelial cells when injected in to ischemic tissue. MicroRNA expression profiling and confirmatory tests indicated that CD34 Exo is significantly enriched with pro-angiogenic miRNAs such as miR126. Treatment of CD34 Exo increased miR126 expression specifically in endothelial cells of ischemic tissue; treatment of CD34 Exo lacking miR126 abolished that increase and diminished its pro-angiogenic function. This, and studies using fluorescent miR126 confirm that miR126 was directly transferred from CD34 Exo to endothelial cells, and that indirect increase of miR126 by other components of CD34 Exo was minimal. This data also suggests that miR126 is important for CD34 Exo function. Ongoing studies will test the role of transferred miR126 on modulation of proangiogenic gene expression pathways in endothelial cells lacking miR126 either by dicer, or, Egfl-7 knockdown. Conclusion: CD34 + exosomes transfer miR126 to endothelial cells to induce angiogenesis and myocardial repair. The functional benefits associated with CD34 + cell therapy may be mediated by exosomes-mediated transfer of angiogenic microRNAs to endothelial cells.