RAS P21 Protein Activator Research Articles

DNA Methylation of BDNF and RASA2 Genes Is Associated With Cognitive Function in Postmenopausal Women With Breast Cancer.

To determine associations among DNA methylation of brain-derived neurotrophic factor (BDNF) and RAS p21 protein activator 2 (RASA2) genes with processing speed and perceived cognitive function. This was a cross-sectional, secondary analysis of baseline data from a randomized controlled trial, the Exercise Program in Cancer and Cognition Study. Data included M values for DNA methylation of the BDNF and RASA2 genes; processing speed, objectively measured using the Grooved Pegboard and Digit Vigilance Test scores; and perceived cognitive function, self-reported using the Patient Assessment of Own Functioning Inventory. Regression analysis was conducted. Greater methylation of cg21291635 of the BDNF gene (p = 0.01) and cg20247102 of the RASA2 gene (p = 0.013) were associated with poorer processing speed, whereas greater methylation of cg20108357 of the BDNF gene (p < 0.001) and cg00567892 of the RASA2 gene (p = 0.019) were associated with better perceived cognitive function. Gene methylation variations were demonstrated, suggesting the genes' potential roles and two possible distinct mechanisms of cognitive function in cancer.&nbsp.

Imaging Assessment of Fetal Cerebrovascular Anomalies.

Four distinct vascular anomalies can be seen to affect the brain on fetal imaging: vein of Galen malformations, non-galenic arteriovenous pial fistulas, dural sinus malformations, and intracranial venous malformations. These congenital disorders affect the arteries and veins of the developing brain and are rarely seen beyond the neonatal stage. The four fetal cerebrovascular anomalies are associated with quite disparate natural histories and prognoses. MRI plays a pivotal role in the evaluation of fetuses with these conditions due to its ability to definitively establish the diagnosis, to detect subtle parenchymal injuries, to delineate the course of abnormal vessels in detail and to some extent the nature of vascular flow, and to identify ischemic, thrombotic, and hemorrhagic complications. Recently, an investigational transurterine embolization procedure targeted at treating fetuses with vein of Galen malformations who are at high risk for neonatal decompensation has emerged as a promising alternative to expectant management and post-natal embolization, with imaging being used to identify suitable patients for the intervention and in pre-procedural planning. This manuscript reviews the essential imaging and clinical features of these four fetal neurovascular anomalies and underscores the practical aspects related to counseling, prognosis, and the multidisciplinary management of these entities.ABBREVIATIONS: ACVRL1= activin A receptor like type 1; b-SSFP=Balanced Steady State Free precession; DSM= Dural Sinus Malformation; Ephrin B4= Ephrin type-B receptor 4; icVM= Intracranial Venous Malformation; ITGB1= Integrin Subunit Beta 1; NOTCH1= Neurogenic locus notch homolog protein 1; PTPN11= Protein Tyrosine Phosphatase Non-Receptor Type 11; RASA1= RAS P21 Protein Activator 1; SSFSE= Single-shot fast spin echo; VOGM=Vein of Galen Malformation.

The role of RASA2 in predicting radioresistance in lung cancer through regulation of p53.

One of the most common causes of lung cancer relapse after clinical treatment is radioresistance. However, the mechanism underlying radioresistance remains unclear. In this study, we investigated the role of Ras p21 protein activator (RASA2) in non-small cell lung cancer (NSCLC). The messenger RNA (mRNA) of RASA2 was tested via reverse-transcription quantitative polymerase chain reaction (RT-qPCR) of cancer tissues from patients with NSCLC. Computed tomography (CT) and bioluminescent imaging (BLI) were used to monitor the tumor growth of patients and orthotopic mice, respectively. Protein-protein interaction was quantified via immunoprecipitation and glutathione S transferase (GST) pulldown assay. Western blotting was used to evaluate the phosphorylation and ubiquitination level of p53. The results indicated a negative correlation between the mRNA expression levels of RASA2 in tumor tissues with patients' response to radiotherapy. Patients with a high expression of RASA2 had a lower objective response rate (ORR) after 1 month of radiotherapy than patients with low expression of RASA2 after 1 month of radiotherapy. In terms of mechanism, we proved that RASA2 can directly bind to p53 to promote the phosphorylation of p53, which inhibits its transcriptional activity and further promotes its degradation through the ubiquitin/proteasome pathway. In this process, the apoptosis of tumor cells is inhibited due to impaired p53 surveillance, which leads to radioresistance. Our results demonstrate that RASA2 negatively regulates p53 in cancer cells and therefore promotes radioresistance, providing a new predictive biomarker and a potential therapeutic target for radioresistance.

Kechuanning Gel Plaster Exerts Anti-inflammatory and Immunomodulatory Effects on Ovalbumin-induced Asthma Model Rats via ERK Pathway.

We aimed to evaluate the therapeutic effects of Kechuanning gel plaster on ovalbumin (OVA)-induced rat model of asthma. Rats were injected with OVA to induce asthma, and Kechuanning gel plaster was administered after the OVA challenge. The immune cell counts in the bronchial alveolar lavage fluid (BALF) were calculated after Kechuanning gel plaster administration. The levels of immune factors in BALF and serum OVA-specific IgE levels were analyzed. Western blot analysis and immunohistochemistry were carried out to analyze the following proteins: C-FOS, C-JUN, RAS p21 protein activator 1 (RASA1), matrix metalloproteinase 9 (MMP9), RAF1, p-MEK1, tissue inhibitor of metalloproteinase-1 (TIMP1), and p-extracellular signal-regulated kinase 1 (ERK1). Administration of Kechuanning gel plaster led to decreased immune cell counts, inflammatory cytokines (interleukin (IL)-1β, IL13, and IL17), and OVA-specific IgE expression. Compared to the normal group, the C-FOS, C-JUN, RASA1, MMP9, RAF1, MEK1, TIMP1, and p- ERK1 expressions in the model group were significantly increased, whereas Kechuanning gel plaster administration decreased C-JUN, MMP9, TIMP1, RAF1, MEK1, p-ERK1, C-FOS, and RASA1 protein levels. Kechuanning gel plaster exerted its therapeutic effects on OVA-induced asthma model rats through the ERK signaling pathway. Kechuanning gel plaster could be considered as a potential alternative therapeutic agent for the management of asthma.

MiR-182/183-Rasa1 axis induced macrophage polarization and redox regulation promotes repair after ischemic cardiac injury

Few therapies have produced significant improvement in cardiac structure and function after ischemic cardiac injury (ICI). Our possible explanation is activation of local inflammatory responses negatively impact the cardiac repair process following ischemic injury. Factors that can alter immune response, including significantly altered cytokine levels in plasma and polarization of macrophages and T cells towards a pro-reparative phenotype in the myocardium post-MI is a valid strategy for reducing infarct size and damage after myocardial injury.Our previous studies showed that cortical bone stem cells (CBSCs) possess reparative effects after ICI. In our current study, we have identified that the beneficial effects of CBSCs appear to be mediated by miRNA in their extracellular vesicles (CBSC-EV). Our studies showed that CBSC-EV treated animals demonstrated reduced scar size, attenuated structural remodeling, and improved cardiac function versus saline treated animals. These effects were linked to the alteration of immune response, with significantly altered cytokine levels in plasma, and polarization of macrophages and T cells towards a pro-reparative phenotype in the myocardium post-MI. Our detailed in vitro studies demonstrated that CBSC-EV are enriched in miR-182/183 that mediates the pro-reparative polarization and metabolic reprogramming in macrophages, including enhanced OXPHOS rate and reduced ROS, via Ras p21 protein activator 1 (RASA1) axis under Lipopolysaccharides (LPS) stimulation. In summary, CBSC-EV deliver unique molecular cargoes, such as enriched miR-182/183, that modulate the immune response after ICI by regulating macrophage polarization and metabolic reprogramming to enhance repair.

MEG3 sponges miRNA-376a and YBX1 to regulate angiogenesis in ovarian cancer endothelial cells

ObjectivesRecent studies have demonstrated maternally expressed gene 3 (MEG3) as a tumor suppressor across multiple malignancies. Meanwhile, the role of MEG3 in ovarian cancer needs further investigation. We aim to study the effects of MEG3 on angiogenesis in ovarian cancer and the underlying mechanisms. MethodsThe transcript levels of MEG3 in ovarian cancer samples from the GEPIA database were analyzed and compared to those in normal samples. The effect of MEG3 on the tube formation ability was quantified in ovarian carcinoma-derived microvascular endothelial cells (ODMECs). Through sequence analysis, we identified miR-376a as a major candidate to bind to MEG3. A MEG3-miR-376a binding site was identified via genetic modulation methods. RAS p21 protein activator 1 (RASA1) was screened as a middle player to bridge the role of miR-376a and angiogenesis. The regulation between miR-376a and RASA1 was confirmed via a dual-luciferase reporter assay. Finally, the competition was explored between Y-box binding protein 1 (YBX1) and miR-376a in binding to MEG3. ResultsMEG3 was significantly downregulated in ODMECs compared with normal ovarian endothelial cells. Overexpression of MEG3 led to reduced tube formation of ODMECs. The MS2 hairpin assay showed that MEG3 acted as a platform to sponge miR-376a. RASA1, a key suppressor of tube formation, was directly targeted by miR-376a. Further, MEG3 suppressed angiogenesis through the miR-376a/RASA1 axis in ODMECs. Finally, YBX1 and miR-376a were competitively bound to MEG3. ConclusionThis study uncovered a novel mechanism that MEG3 sponged miRNA-376a and YBX1 to regulate the expression of RASA1 and exert an effect on the angiogenesis of ovarian cancer.

EPHB4-RASA1-Mediated Negative Regulation of Ras-MAPK Signaling in the Vasculature: Implications for the Treatment of EPHB4- and RASA1-Related Vascular Anomalies in Humans.

Ephrin receptors constitute a large family of receptor tyrosine kinases in mammals that through interaction with cell surface-anchored ephrin ligands regulate multiple different cellular responses in numerous cell types and tissues. In the cardiovascular system, studies performed in vitro and in vivo have pointed to a critical role for Ephrin receptor B4 (EPHB4) as a regulator of blood and lymphatic vascular development and function. However, in this role, EPHB4 appears to act not as a classical growth factor receptor but instead functions to dampen the activation of the Ras-mitogen activated protein signaling (MAPK) pathway induced by other growth factor receptors in endothelial cells (EC). To inhibit the Ras-MAPK pathway, EPHB4 interacts functionally with Ras p21 protein activator 1 (RASA1) also known as p120 Ras GTPase-activating protein. Here, we review the evidence for an inhibitory role for an EPHB4-RASA1 interface in EC. We further discuss the mechanisms by which loss of EPHB4-RASA1 signaling in EC leads to blood and lymphatic vascular abnormalities in mice and the implications of these findings for an understanding of the pathogenesis of vascular anomalies in humans caused by mutations in EPHB4 and RASA1 genes. Last, we provide insights into possible means of drug therapy for EPHB4- and RASA1-related vascular anomalies.

Enhanced Apoptosis in Choroidal Tissues in Lens-Induced Myopia Guinea Pigs by Activating the RASA1 Signaling Pathway.

PurposeThis study aimed to explore the role of the RAS p21 protein activator 1 (RASA1) signaling pathway in apoptosis in choroid tissues from guinea pigs with negative lens-induced myopia (LIM).MethodsBiometric measurements were performed to examine refractive status, ocular parameters, and choroidal thickness (ChT) after myopia induction. The choroidal morphology was observed by hematoxylin and eosin (H&E) staining and TUNEL assay. The expression of the RASA1 signaling pathway at the mRNA and protein levels in choroidal tissues was measured by real-time quantitative PCR (qPCR) and western blot assays.ResultsCompared with the normal control (NC) group, the ocular length of the guinea pigs in LIM increased remarkably, as did the myopic refraction. ChT decreased after myopia induction. H&E staining showed that the thickness and laxity of the choroidal tissues in LIM were strikingly reduced. The number of apoptotic cells in the LIM eyes was increased. Moreover, qPCR and western blot assays showed that the expression levels of both RASA1 and BCL-2-associated agonist of cell death (BAD) were higher in the LIM group than in the NC group, whereas the expression level of B-cell lymphoma 2 (BCL-2) was decreased after 2 weeks of experimental myopia. However, the trend of RASA1, BAD, and BCL-2 expression was reversed after 4 weeks of experimental myopia compared with levels after 2 weeks of experimental myopia.ConclusionsResults showed that the RASA1 signaling pathway is activated in choroid tissues in myopic guinea pigs. Activated RASA1 signaling induces high BAD expression and low BCL-2 expression, which in turn promotes apoptosis and ultimately causes ChT thinning in myopic guinea pigs.

A Sampling of Highlights from the Literature.

Knockout of RASA2 enhances T-cell antitumor function (from Public Domain Vectors)Several immunosuppressive mechanisms limit the efficacy of adoptive T-cell therapies. Carnevale et al. performed six genome-wide CRISPR–Cas9 knock-out screens for regulators of T-cell resistance to immunosuppression by inhibitors of calcium and calcineurin, TGFβ, and regulatory T cells, identifying a role for RAS p21 protein activator 2 (RASA2) in each case. Knocking out RASA2A in human T cells, including T-cell receptor (TCR) and chimeric antigen receptor (CAR)–engineered T cells, enhances their antitumor function in vitro and in xenograft models of melanoma, osteosarcoma, and acute lymphoblastic leukemia. The data suggest ablation of RASA2 may enable the generation of more efficacious adoptive T-cell therapies.Carnevale J, …, Marson A. Nature 2022 August 24;609:174–82.CD62L+ TPEX cells mediate responses to anti–PD-1 (from NIAID via NIH Flickr)Precursors of exhausted T (TPEX) cells, characterized by expression of TCF1, mediate responsiveness to anti–PD-1. Following single-cell RNA sequencing of TPEX cell–enriched CD8+ T cells from mice chronically infected with lymphocytic choriomeningitis virus (LCMV), Tsui et al. stratify TPEX cells by expression of Sell, which encodes CD62L. The transcription factor MYB is required for the development of CD62L+ TPEX cells, which show enhanced capacity for self-renewal, multipotency, and long-term proliferation compared with CD62L– TPEX cells. CD62L+ TPEX cells are pivotal for responsiveness to anti–PD-1 in mice chronically infected with LCMV. Thus, it is vital that we better understand their role in T cell–mediated cancer immunotherapy.Tsui C, …, Kallies A. Nature 2022 August 17;609:354–60.iNKT cells outperform other cell therapies (from Todd van Hoosear via Flickr)The immune suppressive tumor microenvironment (TME) can curb responses to an array of cell-based therapies. Delfanti et al. demonstrate the utility of T-cell receptor (TCR)–engineered invariant natural killer T (iNKT) cells to overcome immune-suppressive barriers in the TME. The engineered iNKT cells act as bispecific effectors, bridging CD1d to MHC-restricted antigens on tumor cells, while also maintaining their known TME-modulating functions. Treatment with these cells outperforms nontransduced iNKT cells and CD8+ T cells expressing the same TCR, and efficacy can be boosted with addition of an iNKT-cell agonist. These data show that TCR-engineered iNKT cells could be a potential cell-based therapy for patients with cancer.Delfanti G, …, Casorati G. Sci Immunol 2022 August 19;7:sciimmunol.abn65.Targeting COX2+ fibroblasts can improve antitumor responses (from go digital via Flickr).How the premetastatic niche develops and what cell types in the metastasis-supporting organ are involved has not been fully elucidated. Gong et al. identify a subset of cyclooxygenase 2–positive (COX-2+) lung fibroblasts that modulate the function of multiple myeloid cell subsets, rendering them dysfunctional or immune suppressive. This process is enhanced by neutrophil-derived IL1β produced because of tumor-associated inflammation. In breast cancer models, targeting the COX2 pathway in these fibroblasts improves immunotherapy efficacy and reduces lung metastasis, highlighting how understanding the microenvironment of the premetastatic niche can facilitate identification of therapeutic targets to boost antitumor responses.Gong Z, …, Ren G. Immunity 2022 July 30;55:1483–1500.E9.In situ generation of CAR-macrophages effectively prevent GBM recurrence (from Christaras A via Wikimedia Commons)Recurrence of glioblastoma multiforme (GBM) after surgical resection is driven by residual glioma stem cells (GSC). Chen et al. developed a cavity-injectable hydrogel embedded with macrophage-targeted nanoporter particles that they used to deliver plasmids encoding a CD133-directed chimeric antigen receptor (CAR) to macrophages in situ following tumor resection in preclinical models. The CAR+ macrophages target GSCs and show antitumor activity in vitro and in vivo, including preventing recurrence of surgically resected GBM in an orthotopic patient-derived xenograft model. These findings suggest that the injectable CAR-macrophage platform may be an effective therapeutic strategy to improve long-term survival of GBM patients.Chen C, …, Jiang X. Sci Trans Med 2022 Aug 3;14:scitranslmed.abn1128.Tumor-reactive T cells exhibit signatures of activation indicative of their TCR avidity (from Takuma-sa via Wikimedia Commons)The impact of T-cell receptor (TCR) avidity on T-cell antitumor activity remains unclear. Through adoptive transfer of ultra-low (<1000) numbers of T cells with OVA-specific TCRs of varying avidity into mice bearing OVA-expressing tumors, Purcarea et al. found that tumor-reactive T cells in tissues outside of tumors express PD-1 proportional to the avidity of their TCR and to their protective capacity. Tumor-infiltrating and peripheral T cells isolated from human melanoma patients exhibit gene expression and phenotypic signatures of ex vivo TCR functionality and cytotoxicity, altogether presenting molecular markers of highly active tumor-specific T cells.Purcarea A, …, Schober K. Sci Immunol 2022 August 12;7:sciimmunol.abm207.

MicroRNA-4487 regulates vascular smooth muscle cell proliferation, migration and apoptosis by targeting RAS p21 protein activator 1

BackgroundDysregulation of microRNA (miRNA) is involved in the pathogenesis of a variety of diseases, including atherosclerosis (AS). However, the role of miRNA-4487 (miR-4487) in the development of AS is not fully clarified. This study is intended to investigate the regulatory effects of miR-4487 on the proliferation, migration and apoptosis of vascular smooth muscle cells (VSMCs) and the related mechanisms. MethodsOxidized low-density lipoprotein (ox-LDL) was employed to induce the dysfunction of VSMCs. Subsequently, miR-4487 expression was detected by quantitative real-time PCR. Afterward, the expression levels of RAS p21 protein activator 1 (RASA1) and apoptosis-related proteins (Bcl-2, Bax, Cleaved caspase 3, Cleaved caspase 9) were detected by Western blotting. The proliferation, migration and apoptosis of VSMCs were then detected by CCK-8, BrdU, Transwell and flow cytometry assays, respectively. Moreover, a dual-luciferase reporter gene assay was executed to verify the targeting between miR-4487 to the RASA1 3′-untranslated region (3′-UTR). Resultsox-LDL treatment increased miR-4487 expression and decreased RASA1 expression in VSMCs. Additionally, ox-LDL treatment promoted the proliferation and migration of VSMCs, but inhibited apoptosis. Besides, the effects of ox-LDL treatment on the proliferation, migration and apoptosis of VSMCs were attenuated by the transfection of miR-4487 inhibitors. Furthermore, miR-4487 directly targeted the 3′-UTR of RASA1 mRNA and repressed the expression level of RASA1. Also, RASA1 knockdown reversed the effects of miR-4487 inhibition on VSMCs. ConclusionMiR-4487 promotes VSMCs viability and migration and inhibits apoptosis by targeting RASA1 in VSMCs, by which it promotes the pathogenesis of AS.

Prothymosin α Gene Transfer Modulates Myocardial Remodeling after Ischemia-Reperfusion Injury.

Prothymosin α (ProT), a polypeptide, attenuates inflammation and inhibits transforming growth factor (TGF)-β signaling in pulmonary tissues. We investigated the potential role of ProT in myocardial ischemia-reperfusion (MyoIR) injury using ProT cDNA transfer. Serum ProT levels were investigated in cardiogenic shock patients with MyoIR (n = 9). In addition, the myocardium of Sprague-Dawley rats (n = 52) was subjected to 25 min of ischemia followed by an injection of adenoviral vectors (2 × 109 plaque-forming units) carrying ProT or the luciferase gene, 10 min before reperfusion. Echocardiography, serum ProT, and biochemical analyses of organ functions were performed before euthanasia, 14 days after treatment. Immunohistochemistry and immunoblotting of the myocardial tissue were also performed. Serum ProT levels were transiently elevated in the rats and patients early after MyoIR, which was reduced to baseline levels in control rats and patients. ProT gene transfer persistently mobilized ProT serum levels, reduced dilatation, attenuated fibrotic changes, and preserved the left ventricular ejection fraction after MyoIR. Tissue thrombospondin-1 level was abundant, and matrix metalloproteinase-2, collagen I, and collagen IV levels were decreased in the treatment group. While TGF-β protein level remained stable, ProT transduction mobilized Smad7, which counteracted TGF-β. ProT reduced tissue microRNA-223 expression, inhibited the associated interleukin-1β, and preserved RAS p21 protein activator 1 protein abundance. An increase in transient serum ProT levels could be a protective response in the acute stage of MyoIR. ProT gene transfer further preserved ventricular morphology and function through anti-inflammatory and anti-fibrotic effects in the subacute stage after injury.

New Vistas in microRNA Regulatory Interactome in Neuropathic Pain.

Neuropathic pain is a chronic pain condition seen in patients with diabetic neuropathy, cancer chemotherapy-induced neuropathy, idiopathic neuropathy as well as other diseases affecting the nervous system. Only a small percentage of people with neuropathic pain benefit from current medications. The complexity of the disease, poor identification/lack of diagnostic and prognostic markers limit current strategies for the management of neuropathic pain. Multiple genes and pathways involved in human diseases can be regulated by microRNA (miRNA) which are small non-coding RNA. Several miRNAs are found to be dysregulated in neuropathic pain. These miRNAs regulate expression of various genes associated with neuroinflammation and pain, thus, regulating neuropathic pain. Some of these key players include adenylate cyclase (Ac9), toll-like receptor 8 (Tlr8), suppressor of cytokine signaling 3 (Socs3), signal transducer and activator of transcription 3 (Stat3) and RAS p21 protein activator 1 (Rasa1). With advancements in high-throughput technology and better computational power available for research in present-day pharmacology, biomarker discovery has entered a very exciting phase. We dissect the architecture of miRNA biological networks encompassing both human and rodent microRNAs involved in the development of neuropathic pain. We delineate various microRNAs, and their targets, that may likely serve as potential biomarkers for diagnosis, prognosis, and therapeutic intervention in neuropathic pain. miRNAs mediate their effects in neuropathic pain by signal transduction through IRAK/TRAF6, TLR4/NF-κB, TXIP/NLRP3 inflammasome, MAP Kinase, TGFβ and TLR5 signaling pathways. Taken together, the elucidation of the landscape of signature miRNA regulatory networks in neuropathic pain will facilitate the discovery of novel miRNA/target biomarkers for more effective management of neuropathic pain.

Regulation of IncRNA ZNF667-AS1 in Proliferation and Invasion of Esophageal Squamous Cell Carcinoma Cells via Mediating ceRNA Network.

Esophageal squamous cell carcinoma (ESCC), classified as a primary histological subtype of esophageal cancer (EC), dominates approximately 90% of the newly diagnosed EC. Long non-coding RNAs (lncRNAs) are frequently related to the course of ESCC. The current study aimed to investigate whether lncRNA zinc finger protein 667-antisense RNA 1 (ZNF667-AS1) modulates the proliferation and invasion of ESCC cells. ESCC tissues and cell lines, para-carcinoma tissues, and human esophageal epithelial cells (HEEpiCs) were collected. lncRNA ZNF667-AS1 expression in the above tissues and cells was detected. The effect of lncRNA ZNF667-AS1 on proliferation and invasion of Eca109 cells was detected using cell counting kit-8, colony formation, and Transwell assays. lncRNA ZNF667-AS1 subcellular localization was determined via the nuclear/cytosol fractionation assay. The binding relationships between miR-18b-5p and lncRNA ZNF667-AS1 and RAS p21 protein activator 1 (RASA1) were verified using dual-luciferase reporter gene experiment and RNA immunoprecipitation experiment. The expressions of miR-18b-5p and RASA1 in the tissues and cells were identified. The roles of miR-18b-5p overexpression or silencing RASA1 in proliferation and invasion of ESCC cells were examined through rescue experiments. lncRNA ZNF667-AS1 was underexpressed in ESCC tissues and cells, and lncRNA ZNF667-AS1 overexpression hampered ESCC cell proliferation and invasiveness. miR-18b-5p targeted RASA1 while lncRNA ZNF667-AS1 promoted RASA1 transcription via binding to miR-18b-5p. Over-expression miR-18b-5p or silencing RASA1 reversed the inhibitory effects of lncRNA ZNF667-AS1 overexpression on ESCC cell proliferation and invasion. lncRNA ZNF667-AS1 overexpression accelerated RASA1 transcription by competitively binding to miR-18b-5p, thus suppressing ESCC cell proliferation and invasion.

Identification of the circ_PRKDC/miR-20a-3p/RASA1 axis in regulating HaCaT keratinocyte migration.

Migration of keratinocytes plays a crucial role in the re-epithelialization phase during wound healing. Circular RNA (circRNA) protein kinase, DNA-activated, catalytic subunit (circ_PRKDC, hsa_circ_0084443) has been identified as a regulator of keratinocyte migration. However, the molecular basis governing it remains unclear. The levels of circ_PRKDC, microRNA (miR)-20a-3p, and RAS p21 protein activator 1 (RASA1) were assessed by quantitative real-time PCR (qRT-PCR) or western blot. Subcellular localization, Actinomycin D, and Ribonuclease (RNase) R assays were performed to characterise circ_PRKDC. Cell migration was gauged by transwell and wound-healing assays. A direct relationship between miR-20a-3p and circ_PRKDC or RASA1 was verified by dual-luciferase reporter and RNA pull-down assays. Circ_PRKDC expression was reduced in wound skin during wound healing. Circ_PRKDC modulated migration of HaCaT keratinocytes. Mechanistically, circ_PRKDC directly targeted miR-20a-3p. The regulation of circ_PRKDC on HaCaT keratinocyte migration was mediated by miR-20a-3p. RASA1 was identified as a direct and functional target of miR-20a-3p, and miR-20a-3p-mediated inhibition of RASA1 impacted HaCaT keratinocyte migration. Circ_PRKDC acted as a post-transcriptional modulator of RASA1 expression through miR-20a-3p. Moreover, circ_PRKDC modulated migration of HaCaT keratinocytes by RASA1. Our findings demonstrated a novel molecular basis, the miR-20a-3p/RASA1 axis, for the regulation of circ_PRKDC on HaCaT keratinocyte migration.

RASA4 inhibits the HIFα signaling pathway to suppress proliferation of cervical cancer cells

ABSTRACT RAS p21 protein activator 4 (RASA4) has been recognized as a Ca2+-promoted Ras–MAPK pathway suppressor that inhibits tumor growth. However, the role of RASA4 in cervical squamous cell carcinoma (CESC) remains unclear. The mRNA levels of RASA4 were analyzed using the GEO and GEPIA databases. Kaplan–Meier analysis and ROC analyses were conducted to determine the prognostic and diagnostic values for patients from the TCGA-CSCE cohort. The CCK8 and colony assays were performed to assess the impact of RASA4 ectopic expression and gene inactivation on tumor cell proliferation. In vivo experiments were performed. Luciferase reporter assays and LW6 (a HIFα inhibitor) were employed to verify the regulatory relationship between RASA4 and the HIFa signaling pathway. The GEPIA and GEO database analysis demonstrated poorly expressed RASA4 in the CESC tissues relative to that in the noncancerous tissues. Based on the TCGA database, poorly expressed RASA4 signified high prognostic and diagnostic values. Ectopically expressed RASA4 weakened the proliferative potential of HeLa cells, whereas RASA4 genetic inactivation produced the opposite impact in the HeLa and C-33A cells. The promoting effect of RASA4 deficiency on tumourigenesis was also recorded in vivo. Subsequently, RASA4 negatively regulated the HIFα-driven luciferase activities and weakened the expression of survivin. Meanwhile, LW6 treatment abrogated the increased proliferation of HeLa cells, as well as the increased expression of survivin by RASA4 depletion. Our findings indicated that RASA4 can inhibit the proliferation of cervical cancer cells by inactivating the HIFα signaling pathway, suggesting novel prospects for targeted therapy against CESC.

A DNA Methylation-Based Gene Signature Can Predict Triple-Negative Breast Cancer Diagnosis.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype and lacks targeted treatment. It is diagnosed by the absence of immunohistochemical expression of several biomarkers, but this method still displays some interlaboratory variability. DNA methylome aberrations are common in BC, thereby methylation profiling could provide the identification of accurate TNBC diagnosis biomarkers. Here, we generated a signature of differentially methylated probes with class prediction ability between 5 non-neoplastic breast and 7 TNBC tissues (error rate = 0.083). The robustness of this signature was corroborated in larger cohorts of additional 58 non-neoplastic breast, 93 TNBC, and 150 BC samples from the Gene Expression Omnibus repository, where it yielded an error rate of 0.006. Furthermore, we validated by pyrosequencing the hypomethylation of three out of 34 selected probes (FLJ43663, PBX Homeobox 1 (PBX1), and RAS P21 protein activator 3 (RASA3) in 51 TNBC, even at early stages of the disease. Finally, we found significantly lower methylation levels of FLJ43663 in cell free-DNA from the plasma of six TNBC patients than in 15 healthy donors. In conclusion, we report a novel DNA methylation signature with potential predictive value for TNBC diagnosis.

The long noncoding RNA MEG3 regulates Ras-MAPK pathway through RASA1 in trophoblast and is associated with unexplained recurrent spontaneous abortion

BackgroundMaternally Expressed Gene 3 (MEG3) is expressed at low levels in placental villi during preeclampsia; however, its roles in unexplained recurrent spontaneous abortion (URSA) remain unclear. In this study, we aimed to explore the relationship between MEG3 and URSA.MethodsThe differentially expressed lncRNAs (MEG3) and its downstream genes (RASA1) were identified using bioinformatics analysis of Genomic Spatial Event (GSE) database. The expression levels of MEG3 in embryonic villis (with gestational ages of 49–63 days) and primary trophoblasts were determined using quantitative RT-PCR assay. A mouse model of Embryo implantation, Cell Counting Kit-8 (CCK-8), flow cytometry, and Transwell migration assays were performed to determine the implantation, proliferative, apoptotic, and invasive capacities of trophoblast. The level of phosphorylated core proteins in the RAS-MAPK pathway were analyzed using Western blot assay. The mechanisms of MEG3 in the regulation of RASA1 were studied by RNA pulldown, RNA immunoprecipitation (RIP), DNA pulldown, and chromatin immunoprecipitation (ChIP) assays.ResultsMEG3 had a low expression level in embryonic villis of 102 URSA patients compared with those of 102 normal pregnant women. MEG3 could promote proliferation and invasion, inhibit the apoptosis of primary trophoblast of URSA patients (PT-U cells), as well as promote embryo implantation of mouse. Besides, MEG3 also promoted the phosphorylation of rapidly accelerated fibrosarcoma (Raf), mitogen-activated protein kinase kinase (MEK), and extracellular-signal-regulated kinase (ERK) proteins. The results of RNA pull down and RIP assays showed that MEG3 bound with the enhancer of zeste homolog 2 (EZH2). The DNA pulldown assay revealed that MEG3 could bind to the promoter sequence of the RAS P21 Protein Activator 1 (RASA1) gene. Further, the ChIP assay showed that MEG3 promoted the binding of EZH2 to the promoter region of the RASA1 gene.ConclusionsThe inactivation of MEG3 in embryonic villi association with URSA; MEG3 inhibited the expression of RASA1 by mediating the histone methylation of the promoter of RASA1 gene by EZH2, thereby activating the RAS-MAPK pathway and enhancing the proliferative and invasive capacities of trophoblasts.

The Correlation between Gene Expression and DNA Methylation Levels of RAS p21 Protein Activator 3 (RASA3) Gene in Saudi Autistic Children

The potential role of DNA methylation pattern in autism has been provided by revealing the differences in methylation level of multiple genes which are significantly associated with their expression and implicated in ASD pathogenesis. RASA3 is a member of GTPase-activating proteins, RASA3 is highly expressed in brain tissues and can be deregulated by different epigenetic mechanisms. Many studies reported that differentially expressed RASA3 is correlated with its aberrant methylation. Accordingly, this has been suggested that deferentially expression of RASA3 may be correlated with its methylation levels which could play a role in ASD which brought our attention to identify differentially-expressed genes that could be associated with their methylation level of ASD in Saudi population, by performing comparative gene expression of RASA3 then investigate its relation to methylation level.&#x0D; This study was conducted on 18 Saudi autistic children as well as their healthy-control siblings. Relative expression of a candidate gene (RASA3) was measured using RT-qPCR. Furthermore, MethyLight assay was performed to estimate methylation level and evaluate its impact on RASA3 expression. Interestingly, RASA3 expression has found to be dysregulated in ASD cases. In contrast, MethyLight assay result showed no differences in the methylation patterns among ASD cases in the candidate region. However, it remains an open question whether these dysregulations of RASA3 expression could be a biomarker for early screening/detection of some cases which may also suggest a role for RasGAPs in autistic brain function.

Inhibition of lncRNA SNHG8 plays a protective role inhypoxia-ischemia-reoxygenation-induced myocardial injury by regulating miR-335 and RASA1 expression.

Long non-coding (lnc)RNAs serve a role in a number of diseases, including different types of cancer and acute myocardial infarction. The aim of the present study was to investigate the protective role of lncRNA small nucleolar RNA host gene 8 (SNHG8) in hypoxia-ischemia-reoxygenation (HI/R)-induced myocardial injury and its potential mechanism of action. Cell viability, proliferation, creatine kinase myocardial band, cell apoptosis and protein expression levels were determined by Cell Counting Kit-8 assay, EdU assay, ELISA, flow cytometry and western blotting, respectively. The association between SNHG8 and microRNA (miR)-335 was confirmed using a dual-luciferase reporter gene assay. The effects of the miR-335 inhibitor transfections had on increasing apoptosis and decreasing H9C2 cell viability were reversed in cells co-transfected with SNHG8 small interfering (si)RNA. Furthermore, it was found that miR-335 could regulate RAS p21 protein activator 1 (RASA1) expression and that transfection with SNHG8 siRNA downregulated RASA1 expression. Silencing of RASA1 protected against HI/R-induced H9C2 cell injury. However, SNHG8 siRNA did not further reduce apoptosis, demonstrating that SNHG8 may act through RASA1, and RASA1 may mediate the protection of SNHG8 siRNA in HI/R myocardial injury. Thus, inhibition of lncRNA SNHG8 alleviated HI/R-induced myocardial damage by regulating miR-335 and RASA1.

Exosome-transmitted miRNA-335-5p promotes colorectal cancer invasion and metastasis by facilitating EMT via targeting RASA1

Exosomal microRNA (miRNA) secretion has been characterized as a vital factor in intercellular communication among cancer cells. However, little is known about cancer-secreted miRNAs specifically involved in metastasis of colorectal cancer (CRC). Here, we found that exosomes derived from metastatic CRC cell line SW620 promoted migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC cells. The profiling of exosome miRNAs revealed that microRNA (miR)-335-5p was highly expressed in exosomes from metastatic SW620 cells compared to those derived from primary SW480 cells. miR-335-5p was transmitted from metastatic SW620 cells to CRC cells via exosomes and promoted migration, invasion, and EMT of CRC cells. Moreover, exosome-transmitted miRNA-335-5p promotes CRC cell invasion and metastasis by facilitating EMT via targeting RAS p21 protein activator 1 (RASA1). Overexpression of RASA1 abolished the promotive effects of exosomal miR-335-5p on CRC cell migration, invasion, and EMT. Collectively, our data revealed that exosomal miR-335-5p derived from metastatic CRC cells promotes CRC cell invasion and metastasis by facilitating EMT via targeting RASA1, which may serve as a potential therapeutic target for CRC metastasis.