Sprouty-related EVH1 Domain-containing Protein Research Articles

Bioinformatics analysis for the identification of Sprouty-related EVH1 domain-containing protein 3 expression and its clinical significance in thyroid carcinoma

The poorly differentiated thyroid carcinoma (THCA) subtype is associated with an aggressive disease course, a less favorable overall prognosis, and an increased risk of distant organ metastasis. In this study, our objective was to explore the potential utility of the Sprouty-related EVH1 domain-containing protein 3 (SPRED3) as a biomarker for early diagnosis and prognosis in THCA patients. The differentially expressed prognostic-related genes associated with THCA were identified by querying The Cancer Genome Atlas (TCGA) database. The difference in the expression of the SPRED3 gene between thyroid carcinoma (THCA) tissues and normal tissues was analyzed using data from The Cancer Genome Atlas (TCGA) and further validated through immunohistochemistry. Univariate and multivariate Cox regression models were used, along with clinical information from THCA patients, to analyze the prognostic value of the SPRED3 gene in THCA patients. Functional enrichment analysis was subsequently performed to elucidate the molecular mechanisms underlying the regulatory effects of the SPRED3 gene on thyroid carcinoma. Additionally, we calculated the percentage of infiltrating immune cells in THCA patients and evaluated their correlation with SPRED3 gene expression. Compared with those in noncancerous thyroid tissue, the gene and protein expression levels of SPRED3 were found to be elevated in thyroid carcinoma tissues. Furthermore, the expression of SPRED3 in thyroid carcinoma exhibited significant correlations with tumor location, histological grade, pathological stage, and tumor node metastasis classification (TNM) stage. Univariate and multivariate Cox proportional hazards (Cox) regression analyses demonstrated that SPRED3 could serve as an independent prognostic factor for predicting the overall survival of THCA patients. The results of functional enrichment analysis suggested the potential involvement of SPRED3 in the regulation of extracellular matrix organization, epidermal development, signaling receptor activator activity, skin development, receptor ligand activity, glycosaminoglycan binding, neuroactive ligand‒receptor interaction, the IL-17 signaling pathway, and the PI3K-Akt signaling pathway. Additionally, there were significant correlations between the expression level of the SPRED3 gene and the infiltration of various immune cells (eosinophils, central memory T cells, neutrophils, macrophages, and NK cells) within the thyroid tumor microenvironment. SPRED3 can be used as a prognostic biomarker in patients with THCA could potentially be therapeutic target for THCA.

Long noncoding RNA LOC646029 functions as a ceRNA to suppress ovarian cancer progression through the miR-627-3p/SPRED1 axis.

Long noncoding RNAs (lncRNAs) play a crucial regulatory role in the development and progression of multiple cancers. However, the potential mechanism by which lncRNAs affect the recurrence and metastasis of ovarian cancer remains unclear. In the current study, the lncRNA LOC646029 was markedly downregulated in metastatic ovarian tumors compared with primary tumors. Gain- and loss-of-function assays demonstrated that LOC646029 inhibits the proliferation, invasiveness, and metastasis of ovarian cancer cells in vivo and in vitro. Moreover, the downregulation of LOC646029 in metastatic ovarian tumors was strongly correlated with poor prognosis. Mechanistically, LOC646029 served as a miR-627-3p sponge to promote the expression of Sprouty-related EVH1 domain-containing protein 1, which is necessary for suppressing tumor metastasis and inhibiting KRAS signaling. Collectively, our results demonstrated that LOC646029 is involved in the progression and metastasis of ovarian cancer, which may be a potential prognostic biomarker.

Recurrent posterior fossa group A (PFA) ependymoma in a young child with constitutional mismatch repair deficiency (CMMRD).

Recurrent posterior fossa group A (PFA) ependymoma in a young child with constitutional mismatch repair deficiency (CMMRD).

Exosomes derived from stem cells from apical papilla promote angiogenesis via miR-126 under hypoxia.

To explore the effect of exosomal miR-126 derived from stem cells from the apical papilla (SCAPs) under hypoxia on human umbilical vein endothelial cell (HUVEC) angiogenesis. miR-126 mimics plasmids were used to upregulate miR-126 in SCAPs. Internalization of PKH26-labeled exosomes was examined by fluorescent microscopy. CCK-8 assay, Transwell assay, scratch assay, tube formation assay, and Matrigel plug assay were performed to detect the effects of exosomes on the angiogenic ability of HUVECs. The luciferase reporter assay and rescue assay were performed to examine the relationship between miR-126 and sprouty-related, EVH1 domain-containing protein 1 (SPRED1). The involvement of SPRED1 and the extracellular signal-regulated kinase (ERK) signaling pathway was evaluated by western blotting. miR-126 expression was upregulated in SCAPs and in SCAP-derived exosomes under hypoxia. miR-126 expression was increased in HUVECs when cocultured with SCAP-derived exosomes. Induced overexpression of miR-126 in hypoxic SCAPs and secreted exosomes resulted in enhanced angiogenesis both in vitro and in vivo. Western blot analysis revealed that miR-126-mediated SPRED1 downregulation induced activation of ERK signaling. Under hypoxic conditions, exosomes derived from SCAPs can promote HUVEC angiogenesis through expression of miR-126, which subsequently suppresses SPRED1 and activates the ERK signaling pathway.

Construction and transfer of PEI-miRNA-126/210 polyplex into Human Umbilical Vein Endothelial Cells with investigation of its effect on islets survival and function

Background:Type 1 diabetes is an autoimmune disorder characterized by the loss of pancreatic islets. Islet allotransplantation is a potentially beneficial therapeutic approach for diabetes. Islets suffer a variety of cellular insults including ischemia and partial vascular loss during isolation, resulting in a significant reduction in viability prior to transplantation. The present study aimed to investigate the effect of angiogenic microRNA (miRNA)-126 and -210 on islet function and viability in an indirect way. Methods:Poly Ethylenimine (PEI)-miRNA-126 and -210 polyplexes were constructed at various Nitrogen/Phosphate (N/P) ratios. After confirmation by gel retardation and ethidium bromide dye exclusion assay, its cytotoxicity and transfection efficiency were analyzed by MTT and fluorescent assays, respectively. After that, the selected polyplexes were used to transfect Human Umbilical Vein Endothelial Cells (HUVECs) in vitro and were indirectly co-cultured with islet cells for three days. Real-time polymerase chain reaction and enzyme-linked immunoassay were conducted to verify the regulation of target genes and the functionality of the islets. Results:The findings showed that PEI could condense miRNAs at N/P=5. The viability of the HUVECs was decreased by increasing the amount of PEI. Additionally, ployplex-126 and -210 led to a decrease in the expressions of target genes, phosphoinositol-3 kinase regulatory subunit 2, sprouty-related EVH1 domain-containing protein 1, and ephrin-A3 in the islets. Moreover, the expressions of Bax and Bcl2 and their ratio in the treated groups by polyplex-126 and -210 led to better survival and function of the islets, with a higher expression of insulin and response to glucose stimulations. Furthermore, polyplex-210 could downregulate the anti-angiogenic protein, thrombospondin 1, compared to the other groups. Finally, the secretion of C-peptide was higher in polyplex-210 than in the other groups, adjusted for insulin secretion. Conclusion:The results indicated that angiogenic miRNAs could promote the survival and function of islet cells by interacting with their targets.

Methylation of SPRED1: A New Target in Acute Myeloid Leukemia.

Sprouty-related, EVH1 domain-containing protein 1 (SPRED1) has been identified as a novel tumor suppressor gene in acute myeloid leukemia (AML). Previous studies showed that SPRED1 methylation levels were significantly increased in AML patients, making it an interesting candidate for further investigations. To confirm the association of SPRED1 methylation, clinical parameters, and known molecular prognosticators and to identify the impact of methylation level on treatment outcome, we conducted this study in a larger cohort of 75 AML patients. Significantly increased methylation levels of SPRED1 were detected at four of ten CpG units by quantitative high-resolution mass spectrometry-based approach (MassARRAY) in AML patients. Whereas overall survival (OS) and relapse-free survival (RFS) showed no statistical difference between hypermethylation and hypomethylation subgroups, the relationship between methylation level and treatment response was indicated in paired samples from pre- and post-induction. To determine the possible mechanism of SPRED1 methylation in AML, we performed in vitro experiments using THP-1 cells, as the latter showed the highest methylation level (determined by utilizing bisulfite modification) among the three AML cell lines we tested. When treated with 5-AZA and lentivirus transfection, upregulated SPRED1 expression, decreased cell proliferation, increased cell differentiation and apoptosis, and inactivated phosphorylated extracellular signal-regulated kinase (p-ERK) were detected in THP-1 cells. These results show that demethylation of SPRED1 can inhibit the proliferation of AML cells and promote their differentiation and apoptosis, possibly by the ERK pathway. The hypermethylation of SPRED1 is a potential therapeutic target for AML.

Expression of Spred2 in the urothelial tumorigenesis of the urinary bladder.

Aberrant activation of the Ras/Raf/ERK (extracellular-signal-regulated kinase)-MAPK (mitogen-activated protein kinase) pathway is involved in the progression of cancer, including urothelial carcinoma; but the negative regulation remains unclear. In the present study, we investigated pathological expression of Spred2 (Sprouty-related EVH1 domain-containing protein 2), a negative regulator of the Ras/Raf/ERK-MAPK pathway, and the relation to ERK activation and Ki67 index in various categories of 275 urothelial tumors obtained from clinical patients. In situ hybridization demonstrated that Spred2 mRNA was highly expressed in high-grade non-invasive papillary urothelial carcinoma (HGPUC), and the expression was decreased in carcinoma in situ (CIS) and infiltrating urothelial carcinoma (IUC). Immunohistochemically, membranous Spred2 expression, important to interact with Ras/Raf, was preferentially found in HGPUC. Interestingly, membranous Spred2 expression was decreased in CIS and IUC relative to HGPUC, while ERK activation and the expression of the cell proliferation marker Ki67 index were increased. HGPUC with membranous Spred2 expression correlated significantly with lower levels of ERK activation and Ki67 index as compared to those with negative Spred2 expression. Thus, our pathological findings suggest that Spred2 counters cancer progression in non-invasive papillary carcinoma possibly through inhibiting the Ras/Raf/ERK-MAPK pathway, but this regulatory mechanism is lost in cancers with high malignancy. Spred2 appears to be a key regulator in the progression of non-invasive bladder carcinoma.

MiR-126-3p contributes to sorafenib resistance in hepatocellular carcinoma via downregulating SPRED1.

BackgroundSorafenib can prolong the survival of patients with advanced hepatocellular carcinoma (HCC). However, drug resistance remains the main obstacle to improving its efficiency. This study aimed to explore the likely molecular mechanism of sorafenib resistance.MethodsDifferentially expressed microRNAs (miRNAs) related to sorafenib response were analyzed with the Limma package in R software. The expression levels of miR-126-3p and sprouty-related EVH1 domain-containing protein 1 (SPRED1) in HCC cells were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell viability and proliferation were detected with Cell Counting Kit-8 (CCK-8), EdU proliferation, and clone formation assays. Transwell assays were performed to measure cell migration and invasion. TargetScan, MicroRNA Target Prediction Database (miRDB), and StarBase v2.0 were used to predict the targets of miR-126-3p. SPRED1 was confirmed as a target gene of miR-126-3p by dual-luciferase reporter assay and Western blotting. Finally, the in vivo anti-tumor effect of LV-miR-126-3p inhibitor combined with sorafenib was evaluated via subcutaneous tumor models.ResultsHCC cells with high expression of miR-126-3p exhibited increased resistance to sorafenib. The results of bioinformatics analysis and the dual-luciferase reporter assay showed that miR-126-3p directly targeted SPRED1. The sensitivity of HCC cells to sorafenib was markedly enhanced by SPRED1 upregulation. Gain- and loss-of function experiments verified that miR-126-3p induced sorafenib resistance in HCC through downregulating SPRED1. Furthermore, the inhibition of miR-126-3p markedly increased the effectiveness of sorafenib against HCC in vivo. Mechanistically, our results suggested that miR-126-3p promoted sorafenib resistance via targeting SPRED1 and activating the ERK signaling pathway.ConclusionsOur study demonstrates that regulating the miR-126-3p/SPRED1 axis might be a promising strategy for enhancing the antitumor effect of sorafenib in the treatment of HCC.

MicroRNA-126: Dual Role in Angiogenesis Dependent Diseases.

MicroRNA-126, a microRNA implicated in blood vessel integrity and angiogenesis is significantly up/down regulated in different physiological and pathological conditions related to angiogenesis such as cardiovascular formation and angiogenesis dependent diseases. MicroRNA-126 plays a critical role in angiogenesis via regulating the proliferation, differentiation, migration, and apoptosis of angiogenesis related cells such as endothelial cells. The aim of this review is to investigate the molecular mechanisms and the effects of microRNA-126 on the process of angiogenesis in pathophysiological conditions. To conduct this review, related articles published between 2001 and 2019 were collected from the PubMed, Web of Science, Google Scholar, Scopus and Scientific Information Database using search terms such as microRNA-126, angiogenesis, cardiovascular disorders, hypoxia, VEFG-A, endothelial cells, VEGF pathway, and gene silencing. Then, the qualified articles were reviewed. MicroRNA-126 regulates the response of endothelial cells to VEGF, through directly repressing multiple targets, including Sprouty-related EVH1 domain-containing protein 1 (SPRED1) and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-b). MicroRNA-126 -3p and microRNA-126 -5p have cell-type and strandspecific functions and also various targets in angiogenesis that lead to the regulation of angiogenesis via different pathways and consequently diverse responses. MicroRNA-126 can bind to multiple targets and potentially be both positive and negative regulators of gene expression. Thus, microRNA-126 could cause the opposite biological effects depending on the context. As a result, understanding the different cellular pathways through which microRNA-126 regulates angiogenesis in various situations is a critical aspect in the development of novel and effective treatments for diseases with insufficient angiogenesis.

SPRED1 Is Downregulated and a Prognostic Biomarker in Adult Acute Myeloid Leukemia.

We report herein that Sprouty-Related EVH1 Domain-Containing Protein1 (SPRED1) is downregulated and a prognostic biomarker in adult acute myeloid leukemia (AML). We determined mRNA levels of SPRED1 in the bone marrow mononuclear cells from adult patients, including 113 AMLs and 22 acute lymphoblastic leukemias (ALLs), as well as in 37 healthy control subjects. Significantly decreased SPRED1 mRNA expression was found in AML patients comparing to those in ALL patients and healthy controls, which was confirmed by immunocytochemistry analysis of SPRED1 protein and ELISA measurement of serum SPRED1 level. Further analysis demonstrated that SPRED1 expression was significantly higher for most patients at complete remission after induction treatment than at diagnosis. Moreover, SPRED1 expression was significantly downregulated in M2 and M3 types. Non-acute promyelocytic leukemia (non-APL) patients with decreased SPRED1 had significantly lower 2-year progression-free survival and event-free survival rates. In vitro, ectopic overexpression of SPRED1 leads to a decrease of extracellular signal-regulated kinase (ERK) phosphorylation, induction of apoptosis and reduction of proliferation of THP-1 cells. Our findings suggest SPRED1 is not only a predictor of treatment response, but also an independent prognostic factor for non-APL, and targeting Ras- Mitogen-activated protein kinase (MAPK) signaling may be a promising strategy for the treatment of AML with downregulation of SPRED1.

Dynamic Changes in miR-126 Expression in the Hippocampus and Penumbra Following Experimental Transient Global and Focal Cerebral Ischemia–Reperfusion

miR-126 which is considered one of the most important miRNAs for maintaining vascular integrity, plays an important role in neuroprotection after cerebral ischemia-reperfusion (I-R). Moreover, vascular endothelial growth factor A (VEGFA), sprouty-related EVH1 domain-containing protein 1 (SPRED1), and Raf-1 are also involved in physiological processes of vascular endothelial cells (ECs). This study investigated how miR-126 changes with reperfusion time in different brain tissues after global cerebral ischemia and focal cerebral ischemia and examined the underlying mechanism miR-126 involving VEGFA, SPRED1, and Raf-1 after I-R. The results indicated decreases in the levels of miR-126-3p and miR-126-5p expression in mice and gerbils after I-R, consistent with the results after oxygen and glucose deprivation and reperfusion (OGD/R) in PC12 cells. Glial cells were activated as neuronal damage gradually increased after I-R. Inhibition of miR-126-3p exacerbated the OGD/R-induced cell death and reduced cell viability. After miR-126-3p inhibition, the levels of SPRED1 and VEGFA expression were increased, and p-Raf-1 expression was decreased after OGD/R. Moreover, based on the intervention of miR-126-3p inhibition, we found that the expression of p-Raf-1 was significantly increased after the intervention of siSPRED1, while it was not statisticallysignificant after intervention of siVEGFA. The reduction of miR-126 expression after global and focal cerebral ischemia exacerbated neuronal death, which was closely related to increasing the SPRED1 activation and inhibiting the Raf-1 expression.

FLT3-ITD Activates Cytoplasmic Drosha-Dependent Non-Canonical Mechanisms of Mir-155 Biogenesis in Acute Myeloid Leukemia

MiRNAs are small noncoding RNAs that control gene expression by binding to cognate sites in the 3′ untranslated region (3′ UTR) of target messenger RNAs (mRNAs). MiRNAs are transcribed as primary (pri) mRNAs and processed into precursor (pre) miRNAs by nuclear DROSHA before being exported into the cytoplasm via the Exportin 5 (XPO5)/RAN-GTP complex. Once in the cytoplasm, pre-miRNAs are further processed by Dicer into mature miRNAs. The FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common mutations in acute myeloid leukemia (AML) and confers growth and survival advantages to leukemia blasts. Among miRNAs, miR-155 has emerged as one of the most significantly upregulated in the FLT3-ITD+ AML and has been shown to play a pivotal role in uncontrolled blast hyperproliferation and survival. Herein, we report a previously unrecognized activity of FLT3-ITD that leads to deregulation of miR-155 expression by providing the evidence for the existence of FLT3-dependent non-canonical mechanisms of miRNA biogenesis. We initially observed that FLT3-ITD blocked the biogenesis of intronic miRNAs via inhibition of the "gatekeeper" XPO5/RAN-GTP complex that allow nucleus-to-cytoplasm pre-miRNA transport. By using in vitro phosphorylation assay with [32P]-ATP labeling, we demonstrated that FLT3-ITD phosphorylates a Sprouty related EVH1 domain-containing protein 1 (SPRED1), and that phospho-SPRED1 in turn inhibited the XPO5/RAN-GTP complex thereby halting transportation of pre-miRNAs from the nucleus to cytoplasm. This resulted in a decrease of several intronic miRNAs involved in normal hematopoiesis (i.e., miR-29b, miR-181a, miR-146b, miR-126). Accordingly, knocking down of SPRED1 expression in FLT3-ITD+ AML cells resulted in increased production of mature intronic miRNAs. Since the XPO5/RAN-GTP complex is the main gatekeeper for miRNA biogenesis, these results appeared in contradiction with the miR-155 upregulation which has been invariably observed in FLT3-ITD+ AML blasts. Differently from intronic miRNAs, miR-155 is hosted at the genomic site of long non-coding RNA (lnc-RNA) and we therefore hypothesized that it follows a different path of biogenesis. In fact forced expression of FLT3-ITD in lin-Sca1+kit+ (LSK) cells decreased intronic miRNA biogenesis (i.e., miR-126) and increase production of lnc-RNA hosted miRNAs (i.e., miR-155). Accordingly, we demonstrated that upon FLT3-ITD activation, AKT phosphorylated DDX3X, a DEAD-box RNA helicase protein thereby reducing its ability to bind heterogeneous nuclear Ribonucleoprotein U (hnRNP U), a component of the hnRNP complex associated with pre-mRNA splicing. Consequently, in FLT3-ITD cells, BIC-155 splicing is decreased. In fact, we measured significant increased levels of phospho-DDX3X and unspliced BIC-155 in primary FLT3-ITD+ blasts compared with FLT3-ITD- AML blasts. In consistent, DDX3X knock-down (KO) in FLT3-ITD- cells decreased BIC-155 splicing whereas DDX3X re-expression reversed these effects. The excess of unspliced BIC-155 RNA then bound to nuclear RNA export factor 1 (NXF1), a shuttle protein that transports poly A+ RNAs from the nucleus to the cytoplasm. In consistent, NXF1 KO blocked unspliced BIC-155 nucleus-to-cytoplasm transportation. In FLT3-ITD+ blasts, once in the cytoplasm, the unspliced BIC-155 RNA was then processed by cytoplasmic isoforms of DROSHA, as demonstrated using immunostaining, RNA Immunoprecipitation (RIP), and other gain- and loss-of-function experiments. Furthermore, overexpression of cytoplasmic DROSHA but not nuclear DROSHA isoform increased the processing of unspliced BIC-155 to mature miR-155. None of the intronic miRNAs studies interacted with cytoplasmic DROSHA. The bound of BIC-155 RNA with cytoplasmic DROSHA was unique of FLT3-ITD+ blasts, as it was not observed in FLT3-ITD- blasts. Thus, our results indicate a two-fold activity of FLT3-ITD that leads to decreased levels of intronic miRNAs via XPO5/RAN-GTP blockage and upregulation of other lnc-RNA hosted miRNAs via cytoplasmic DROSHA activation. The net result is suppression of intronic miRNAs that participates to the regulation of normal hematopoiesis and upregulation of lnc-RNA-hosted miRNA, especially miR-155 that contribute to aberrant blast hyperproliferation in the FLT3-ITD AML phenotype (Figure 1). Disclosures No relevant conflicts of interest to declare.

Exosomes Derived from miR-126-modified MSCs Promote Angiogenesis and Neurogenesis and Attenuate Apoptosis after Spinal Cord Injury in Rats

Spinal cord injury (SCI) is a devastating neurological event that results in incomplete or complete loss of voluntary motor and sensory function. Until recently, there has been no effective curative strategy for SCI. Our previous study showed that microRNA (miR)-126 promoted angiogenesis and attenuated inflammation after SCI; however, the effect of miR-126-based treatment is limited because of the low efficiency of miR delivery in vivo. Recently, accumulating evidence has indicated that exosomes can serve as a valuable therapeutic vehicle for miR delivery to the central nervous system (CNS). Thus, the present study aimed to investigate whether exosomes derived from mesenchymal stem cells (MSCs) can be used to deliver miR-126 to treat SCI. In this study, we found that MSCs can load miR-126 into secreted exosomes. In a rat model of SCI, exosomes transferred miR-126 to the injured site of the spinal cord, reduced the lesion volume and improved functional recovery after SCI. Additionally, miR-126-loaded exosomes promoted angiogenesis post-SCI. Moreover, the administration of miR-126 exosomes promoted neurogenesis and reduced cell apoptosis after SCI. In vitro, we observed that exosomes derived from miR-126-modified MSCs promoted the angiogenesis and migration of human umbilical venous endothelial cells (HUVECs) by inhibiting the expression of Sprouty-related EVH1 domain-containing protein 1 (SPRED1) and phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2). In conclusion, our study demonstrated that exosomes derived from MSCs transfected with miR-126 may promote angiogenesis and neurogenesis, inhibit apoptosis and promote functional recovery after SCI. These findings suggest that exosomes derived from miR-126-modified MSCs may serve as a novel potential therapeutic strategy for treating SCI.

Pathogenic Mutations Associated with Legius Syndrome Modify the Spred1 Surface and Are Involved in Direct Binding to the Ras Inactivator Neurofibromin

Neurofibromatosis type I (NF1) and Legius syndrome are rare inherited disorders that share diagnostic symptoms including dermal abnormalities like axillary and inguinal freckling and café au lait spots. In addition, patients suffering from NF1 have a demanding risk for the development of severe tumors of the peripheral and central nervous system among other NF1-specific symptoms. NF1 and Legius syndrome are caused by alterations in the NF1 and SPRED1 genes encoding the Ras inhibitors neurofibromin and Spred1 (sprouty related EVH1 domain-containing protein), respectively. Neurofibromin functions as a Ras-specific GTPase-activating protein (Ras-GAP), and Spred1 enhances Ras inactivation by recruiting neurofibromin from the cytosol to membrane-anchored Ras. In a previous study, we mapped the Spred binding site to the GAP-related domain of neurofibromin (NF1-GAP) and identified the GAPex subdomain as critical for Spred1 binding. Here, we characterize the binding site of these proteins in more detail focusing on a mutant Spred1 variant carrying a pathogenic missense mutation (threonine 102 to arginine). Introduction of this mutation, which locates at the N-terminal EVH1 domain of Spred1, weakens the interaction with neurofibromin by about 3 orders of magnitude without perturbing the protein fold, and the binding site of NF1-GAP on the mutant Spred1(EVH1) variant can be identified by NMR spectroscopy. Taken together, our data provide structural insight into the interaction of Spred1 and neurofibromin and characterize the structural or functional consequence of selected patient-derived mutations associated with Legius syndrome.

MicroRNA-126 affects cell apoptosis, proliferation, cell cycle and modulates VEGF/TGF-β levels in pulmonary artery endothelial cells.

In the clinic, therapeutic options for pulmonary arterial hypertension are limited; therefore, investigating the therapeutic strategies and novel therapies is critical for pulmonary arterial hypertension (PAH) treatment. This study aimed to evaluate the role of miRNA-126 (miR-126) and its associated signaling pathways and specific mechanisms for the pathogenesis of PAH. The pulmonary artery endothelial cells (PAECs) were isolated and identified. The miR-126 mimic and miR-126 inhibitor were synthesized. LV-3-miR-126 mimic viral vector and LV-3-miR-126 inhibitor vector were established and infected into pulmonary artery endothelial cells. Expression of sprouty-related EVH1 domain-containing protein 1 (SPRED1), phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) and miR-126 were detected using Real-time PCR (RT-PCR). Cell apoptosis (Annexin V-PE/7-AAD) and proliferation (PKH26) were examined by using FACScan flow cytometry. Vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1) and TGF-β3 levels were evaluated using enzyme-linked immunosorbent assay (ELISA) kits. miR-126 inhibited the endothelial cells related to SPRED1 and PIK3R2 expression. Over-expression of miR-126 significantly inhibited the PAECs apoptosis compared to PAECs and blank LV-3 vector group (p<0.05). miR-126 significantly triggered the PAECs proliferation compared to PAECs and blank LV-3 vector group (p<0.05). In functional analysis, miR-126 mimic significantly increased the cells amounts of S phases compared to PAECs and blank LV-3 vector group (p<0.05). Pre-infection with miR-126 mimic significantly enhanced the levels of VEGF, TGF-β1, and TGF-β3 compared to PAECs and blank LV-3 vector group (p<0.05). miR-126 could affect cell apoptosis, proliferation, cell cycle, and modulate VEGF/TGF-β levels.

Invited commentary

Critical limb ischemia is a complex multifactorial disease with limited treatment options. The pathophysiologic mechanism of critical limb ischemia is one of underlying inflammation elaborated by a variety of cells including neutrophils, macrophages, lymphocytes, and cytokines. More recently, investigation into means of modulating angiogenesis and arteriogenesis through the use of endogenous small noncoding RNAs (microRNAs) has been described. MicroRNAs have been thought to have a role in angiogenesis as well as being modulators of vascular inflammation. The authors of this manuscript have conducted experiments using a mouse model of hindlimb ischemia by femoral ligation and local delivery of microRNA-126 (miR-126) through poly lactic-co-glycolic acid (PLGA) nanoparticles as a vehicle to assess the angiogenic effects of local sustained release of miR-126. They show increased perfusion by laser Doppler imaging and increased capillary and arteriolar density in animals treated with miR-126-loaded PLGA nanoparticles. They believe that miR-126 exerts its effect in part through downstream effectors in the vascular endothelial growth factor receptor pathway through SPRED1. The most important finding of this study is identification of a reliable delivery vehicle for targeted microRNA. As the authors comment, they have not demonstrated what effect, if any, the drug delivery system mediated by PLGA has on the vasculature as no administration of PLGA alone was performed. Further experiments will also be performed on animal models of atherosclerosis to quantify functional end points, including walking distance, to elucidate the clinical relevance of miR-126. This study effectively highlights the complexity of the pathogenesis of limb ischemia and mechanisms used to compensate when the ischemia is acute. A genetic background may not play as important a role as risk factors, including smoking, hypertension, and hyperlipidemia, in the development of limb ischemia. The line of investigation proposed by the authors of this manuscript is important, as studies to date identifying molecules that appear to ameliorate critical limb ischemia have thus far not been effective in mitigating the human disease process. Therapeutic angiogenesis by local sustained release of microRNA-126 using poly lactic-co-glycolic acid nanoparticles in murine hindlimb ischemiaJournal of Vascular SurgeryVol. 68Issue 4PreviewRecent studies demonstrate that microRNAs show promising potential, including angiogenesis, in therapeutic intervention. MicroRNA-126 (miR-126) is reported to regulate angiogenesis by blocking Sprouty-related EVH1 domain-containing protein 1 (SPRED1), an endogenous inhibitor of vascular endothelial cell growth factor. In this study, we investigated the angiogenic effects of the sustained release of miR-126 loaded with poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) in a murine hindlimb ischemia model. Full-Text PDF Open Archive

Icariside II ameliorates endothelial dysfunction by regulating the MAPK pathway via miR-126/SPRED1 in diabetic human cavernous endothelial cells.

AimThe aim of the study was to investigate whether miR-126, a regulator of MAPK signaling via targeting sprouty-related EVH1 domain-containing protein 1 (SPRED1) mRNA, is involved in the process by which icariside II (ICA II) ameliorates endothelial dysfunction in human cavernous endothelial cells (hCECs) exposed to a diabetic-like environment.Materials and methodsPrimary hCECs were isolated and divided into three groups, normal control, diabetes mellitus (DM), and DM treated with ICA II. The cell proliferation and migration abilities of the hCECs were examined. The expression levels of endothelial-related microRNAs and relative target mRNAs (SPRED1, phosphoinositol-3 kinase regulatory subunit 2, and vascular cell adhesion molecule 1) of miR-126 were determined by real-time PCR. The protein expression of endothelial nitric oxide synthase, receptor for advanced glycation end products, and SPRED1, and MAPK signaling activities was determined by Western blot analysis. In addition, miR-126 agomir and antagomir were used for transfection into hCECs to further testify the association between miR-126 and its targeting mRNA SPRED1.ResultshCECs induced with glucose plus advanced glycation end product-BSA showed a significant decrease in endothelial nitric oxide synthase, Ki-67, and miR-126 expression; a downregulated cell migration ability and an increased receptor for advanced glycation end products level. ICA II could partially reverse these changes. SPRED1 mRNA showed a contrary tendency with the miR-126-3p changes. The level of SPRED1 protein increased after the hCECs were induced with glucose plus advanced glycation end product-BSA, and ICA II could rescue its aberrant expression. In addition, the MAPK pathway was downregulated in the hCECs under diabetic conditions, and ICA II could partially enhance its signaling activities. miR-126 was obviously downregulated, and SPRED1 was accordingly upregulated after miR-126 antagomir transfection, while ICA II treatment could recover the expressions of both miR-126 and SPRED1. Moreover, the upregulation of miR-126 and the inhibition of SPRED1 were noticed in the diabetic hCECs by further transfection with miR-126 agomir.ConclusionICA II could ameliorate endothelial dysfunction by regulating the MAPK pathway via miR-126/SPRED1 in hCECs exposed to a diabetic-like environment, and ICA II might be a protective agent for endothelial function in diabetic ED.

Therapeutic angiogenesis by local sustained release of microRNA-126 using poly lactic-co-glycolic acid nanoparticles in murine hindlimb ischemia

ObjectiveRecent studies demonstrate that microRNAs show promising potential, including angiogenesis, in therapeutic intervention. MicroRNA-126 (miR-126) is reported to regulate angiogenesis by blocking Sprouty-related EVH1 domain-containing protein 1 (SPRED1), an endogenous inhibitor of vascular endothelial cell growth factor. In this study, we investigated the angiogenic effects of the sustained release of miR-126 loaded with poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) in a murine hindlimb ischemia model. MethodsWe induced mice hindlimb ischemia through femoral artery excision. We randomly assigned the mice to two groups and performed an intramuscular injection of miR-126-loaded PLGA NPs (miR-126) or scrambled miR-loaded PLGA NPs (control) shortly after induction of ischemia. ResultsThe miR-126 expression levels in the ischemic limb at 3 days after treatment were significantly higher in mice treated with miR-126-loaded PLGA NPs than in those with scrambled miR, indicating the fair efficiency of local miR transduction (control vs miR-126: 0.33 ± 0.12 vs 0.74 ± 0.42; P < .05; n = 6). Laser Doppler perfusion imaging revealed that limb blood flow in mice treated with miR-126-loaded PLGA NPs was significantly higher at 14 days after treatment (sham vs control vs miR-126: 0.62 ± 0.09 vs 0.58 ± 0.05 vs 0.72 ± 0.07; P < .001; n = 12). Immunohistochemical analysis indicated that CD31-positive cell density and α-smooth muscle actin-positive vessel density were significantly higher in miR-126-treated mice (control vs miR-126: 0.33 ± 0.12 vs 0.74 ± 0.42; P < .05; n = 6). SPRED1 messenger RNA expression levels were significantly lower in miR-126-treated mice (control vs miR-126: 1.00 ± 0.05 vs 0.81 ± 0.07; P < .05; n = 6). Western blotting indicated that protein levels of pERK/ERK mediated by SPRED1 were significantly higher in miR-126-treated mice (control vs miR-126: 0.29 ± 0.10 vs 0.54 ± 0.21; P < .05; n = 6). ConclusionsThis study suggests that sustained release of miR-126-loaded PLGA NPs might be an effective method in therapeutic angiogenesis for hindlimb ischemia.

NMR resonance assignments of the EVH1 domain of neurofibromin\u2019s recruitment factor Spred1

Neurofibromin and Sprouty-related EVH1 domain-containing protein 1 (Spred1) both act as negative regulators of the mitogen-activated protein kinase pathway and are associated with the rare diseases Neurofibromatosis type 1 and Legius syndrome, respectively. Spred1 recruits the major GTPase activating protein (GAP) neurofibromin from the cytosol to the membrane in order to inactivate the small G protein Ras. These functions are dependent on the N-terminal EVH1 domain and the C-terminal Sprouty domain of Spred1 whereas the former specifically recognizes the GAP related domain of neurofibromin and the latter is responsible for membrane targeting. Within the GAP domain, Spred1 binding depends on the GAPex portion which is dispensable for Ras inactivation. In a first step towards the characterization of the Neurofibromin Spred1 interface in solution we assigned backbone and side chain 1H, 13C, and 15N chemical shifts of the Spred1 derived EVH1 domain. Our chemical shift data analysis indicate seven consecutive β-strands followed by a C-terminal α-helix which is in agreement with the previously reported crystal structure of Spred1(EVH1). Our data provide a framework for further analysis of the function of patient-derived mutations associated with rare diseases.

MiR-126 Regulation of Angiogenesis in Age-Related Macular Degeneration in CNV Mouse Model.

miR-126 has recently been implicated in modulating angiogenic factors in vascular development. Understandings its biological significance might enable development of therapeutic interventions for diseases like age-related macular degeneration (AMD). We aimed to determine the role of miR-126 in AMD using a laser-induced choroidal neovascularization (CNV) mouse model. CNV was induced by laser photocoagulation in C57BL/6 mice. The CNV mice were transfected with scrambled miR or miR-126 mimic. The expression of miR-126, vascular endothelial growth factor-A (VEGF-A), Kinase insert domain receptor (KDR) and Sprouty-related EVH1 domain-containing protein 1 (SPRED-1) in ocular tissues were analyzed by qPCR and Western blot. The overexpression effects of miR-126 were also proven on human microvascular endothelial cells (HMECs). miR-126 showed a significant decrease in CNV mice (p < 0.05). Both mRNA and protein levels of VEGF-A, KDR and SPRED-1 were upregulated with CNV; these changes were ameliorated by restoration of miR-126 (p < 0.05). CNV was reduced after miR-126 transfection. Transfection of miR-126 reduced the HMECs 2D-capillary-like tube formation (p < 0.01) and migration (p < 0.01). miR-126 has been shown to be a negative modulator of angiogenesis in the eye. All together these results high lights the therapeutic potential of miR-126 suggests that it may contribute as a putative therapeutic target for AMD in humans.