EPB41L3 Inhibits the Progression of Cervical Cancer Via the ERK/p38 MAPK Signaling Pathway.

This study was aimed to uncover the character and potential regulatory mechanism of EPB41L3 in cervical cancer (CC). CC cells were injected into BALB/c nude mice (female) to construct a xenograft tumor model. Real-time quantitative polymerase chain reaction (qRT-PCR) and western blot were performed to evaluate the expression of EPB41L3, ERK/p38 MAPK signal markers in CC tissues and cells. Cell counting kit-8 (CCK-8) and Transwell was applied to analyze the viability, invasion, and migration of CC cell lines. EPB41L3 was substantially decreased both in CC tissues and cells. Cell viability, invasion, and migration of CC cells were reduced by overexpressing EPB41L3. Bioinformatics analysis prerdicted that EPB41L3 was strongly related to the ERK/p38 MAPK pathway. Compared with Ad-nc mice, the volume and weight of tumors and ERK/p38 MAPK signal markers were down-regulated in Ad-EPB41L3 mice. After knocking down EPB41L3 with EPB41L3 siRNA (siEPB41L3), the ERK/p38 MAPK pathway was activated. Moreover, SB203580 treatment reversed the effect of EPB41L3 silencing on the improvement in viability, migration, and invasion of CC cells. EPB41L3 suppresses the progression of CC via activating the ERK/p38 MAPK pathway. EPB41L3 may serve as an effective therapeutic target for CC.

Downregulation of lncRNA EPB41L4A-AS1 promotes gastric cancer cell proliferation, migration and invasion.

BACKGROUND: The incidence of gastric cancer ranks the first among digestive tract tumors in China. However, there are no specific symptoms in the early stage of the tumor and the diagnosis process is complex, so more effective detection methods are very needed. In this study, a novel long noncoding RNA (lncRNA) was introduced as a diagnostic biomarker for gastric cancer, which brought new thinking to the exploration of its pathological mechanism and clinical prediction. METHODS: The level of lncRNA EPB41L4A-AS1 (EPB41L4A-AS1) in gastric cancer serum and cells was verified via real-time quantitative polymerase chain reaction (RT-qPCR). Receiver operating characteristic (ROC) curve was performed based on the EPB41L4A-AS1 level, and the diagnostic possibility of EPB41L4A-AS was analyzed. The chi-square test evaluated the correlation between EPB41L4A-AS expression and clinical information. The cells were cultured and transfected in vitro, and the mediations of abnormal EPB41L4A-AS level on the viability and motility of gastric cancer cells were verified through cell counting kit-8 (CCK-8) and Transwell assay. Furthermore, luciferase activity assay was performed to confirm the sponge molecule microRNA-17-5p (miR-17-5p) of EPB41L4A-AS1. RESULTS: EPB41L4A-AS1 was decreased in gastric cancer, and low EPB41L4A-AS1 level indicated resultful diagnostic value. Overexpression of EPB41L4A-AS1 inhibited the activity of gastric cancer cells, while knockdown of EPB41L4A-AS1 promoted tumor deterioration. EPB41L4A-AS1 directly targeted and regulated the expression ofmiR-17-5p. CONCLUSION: This study elaborated that EPB41L4A-AS1 is lowly expressed in gastric cancer. Silencing EPB41L4A-AS1 was beneficial to cell proliferation, migration, and invasion. EPB41L4A-AS1 provides a new possibility for the diagnosis of gastric cancer patients by targeting miR-17-5p.

Cytoskeletal Protein 4.1R in Health and Diseases.

The protein 4.1R is an essential component of the erythrocyte membrane skeleton, serving as a key structural element and contributing to the regulation of the membrane's physical properties, including mechanical stability and deformability, through its interaction with spectrin-actin. Recent research has uncovered additional roles of 4.1R beyond its function as a linker between the plasma membrane and the membrane skeleton. It has been found to play a crucial role in various biological processes, such as cell fate determination, cell cycle regulation, cell proliferation, and cell motility. Additionally, 4.1R has been implicated in cancer, with numerous studies demonstrating its potential as a diagnostic and prognostic biomarker for tumors. In this review, we provide an updated overview of the gene and protein structure of 4.1R, as well as its cellular functions in both physiological and pathological contexts.

Novel mutation in alpha-spectrin gene in Saudi patients with hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common hereditary hemolytic disorder induced by red blood cell (RBC) membrane defect. This study was undertaken to determine mutations in genes associated with RBC membrane defect in patients with HS such as α-spectrin gene (SPTA1), ß-spectrin gene (SPTB), ankyrin gene (ANK1), band 3 anion transport gene (SLC4A1) and erythrocyte membrane protein band 4.1 gene (EPB41). Blood samples were collected from 23 unrelated patients with HS. Patients were diagnosed according to the guidelines from the British Society for Hematology. All hematological examinations for the determination of RBC abnormalities and osmotic fragility tests were conducted. Genomic DNA were extracted from peripheral blood cells and coding exons of known genes for hereditary spherocytosis were enriched using Roche/KAPA sequence capture technology and sequenced on an Illumina system via next-generation sequencing (NGS). The data showed that most of the HS patients confirmed splenomegaly and showed elevated reticulocytes and abnormal bilirubin values. NGS analysis identified the heterozygous variant c.5501G > A in the exon 39 of SPTA1 gene, resulted in a Trp1834*, which leads to a premature stop codon and subsequent mRNA degradation (nonsense- mediated decay) or truncation in α spectrin. Moreover, our data also revealed conventional mutations in genes SPTB, ANK, SLC4A1 and EBP41 in severe patients of HS. In short, this is the first report that determined a novel mutation c.5501G > A in SPTA1 gene in the Saudi population. To the best of our knowledge, this variant c.5501G > A has not been described in global literature so far. This novel mutation in SPTA1 gene is unique in the Saudi population.

Exploring the Diagnostic Potential of EPB41L3 Methylation in Cervical Cancer and Precancerous Lesions: A Systematic Review and Meta-Analysis.

OBJECTIVE: This meta-analysis aimed to comprehensively evaluate the diagnostic use of erythrocyte membrane protein band 4.1like3 (EPB41L3) methylation detection in cervical cancer (CC) and its precancerous lesions. METHODS: CNKI, Wanfang, Cochrane Library, PubMed, and Ovid databases were searched using a combination of subject headings and free words. Pertinent data were retrieved after screening for inclusion and exclusion criteria, and the quality of the included studies was evaluated using QUADAS-2 criteria. The appropriate software was used for heterogeneity analysis and combined effect size calculation. Additionally, sensitivity analysis was used to evaluate the robustness of the combined results, and meta-regression and subgroup analysis were conducted to investigate the origins of heterogeneity. RESULTS: This meta-analysis included six studies, including 525 healthy individuals, 182 cervical intraepithelial neoplasia 1 (CIN1) samples, 182 CIN2 samples, 281 CIN3 samples, and 226 CC samples. EPB41L3 methylation detection for CIN2 and above lesions demonstrated combined sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio (DOR), and the area under the curve of the comprehensive receiver operating characteristic curve of 0.67, 0.76, 3.19, 0.41, 7.60, and 0.80, respectively; CIN3 and above lesions demonstrated these evaluations at 0.73, 0.84, 4.35, 0.33, 23.94, and 0.90, respectively. Meta-regression analysis revealed that the population, time, sample type, detection method, literature quality, and sample size were not significant sources of heterogeneity affecting the combined diagnostic efficacy of CIN2 and above lesions (p > 0.05). Subgroup analysis revealed higher combined diagnostic values of CIN2 and above lesions in retrospective studies, tissue samples, and Chinese populations, with DORs of 41.03, 14.59, and 13.70, respectively. CONCLUSION: EPB41L3 methylation demonstrated a relatively low diagnostic performance in CC and precancerous lesions. However, it merits further investigation as a potential biomarker. Integrating it with multiple gene detection, human papillomavirus testing, and ThinPrep liquid-based cytology test examination is recommended to explore improved diagnostic strategies for CC and its precancerous lesions.

Expression of EPB41L2 in Cancer-Associated Fibroblasts: Prognostic Implications for Bladder Cancer and Response to Immunotherapy.

BACKGROUND: Immunotherapy response in patients with bladder cancer (BLCA) treated with immune checkpoint inhibitors (ICIs) is variable. The accurate evaluation of immunotherapy efficacy may be facilitated by the tumor microenvironment (TME). Erythrocyte membrane protein band 4.1 like 2 (EPB41L2), a cytoskeletal protein with a regulatory role in the TME was intensively investigated to determine its biological characterization, clinical relevance, and predictive value for immunotherapy in BLCA. METHODS: Comprehensive bioinformatics and statistical analyses were conducted to examine gene expression profile, TME components, immune contexture, molecular features, and prediction of immunotherapy response. Immunohistochemistry (IHC) validated the results of the bioinformatics analysis. Association between immune checkpoint genes (ICGs) and EPB41L2-based risk stratification was validated in the IMvigor210 cohort, and their association with ICI response was assessed. RESULTS: EPB41L2 mRNA levels were decreased in BLCA compared to normal tissue. IHC showed reduced EPB41L2 staining intensity in early BLCA tissue. Nevertheless, elevated EPB41L2 expression was observed in cancer-associated fibroblasts (CAFs) with higher histological grade and pathological stage. High EPB41L2 expression served as a poor prognostic factor for BLCA. Single-cell RNA-seq and further analyses revealed that EPB41L2 was mainly expressed in CAFs and promoted TME remodeling. EPB41L2low/ICGshigh patients showed greater benefit from immunotherapy. Gene mutation analysis revealed a close relationship between EPB41L2 and the frequency of oncogenic mutations, including TP53 and FGFR3. CONCLUSION: Comprehensive analysis and IHC confirmed the upregulation of EPB41L2 in BLCA CAFs and its association with TME remodeling. EPB41L2 and ICG expression were identified as combinatorial biomarkers to predict the response to immunotherapy.

Proximity Interactome Analysis of Super Conserved Receptors Expressed in the Brain Identifies EPB41L2, SLC3A2, and LRBA as Main Partners.

The Super-Conserved Receptors Expressed in the Brain (SREBs) form a subfamily of orphan G protein-coupled receptors, highly conserved in evolution and characterized by a predominant expression in the brain. The signaling pathways activated by these receptors (if any) are presently unclear. Given the strong conservation of their intracellular loops, we used a BioID2 proximity-labeling assay to identify protein partners of SREBs that would interact with these conserved domains. Using streptavidin pull-down followed by mass spectrometry analysis, we identified the amino acid transporter SLC3A2, the AKAP protein LRBA, and the 4.1 protein EPB41L2 as potential interactors of these GPCRs. Using co-immunoprecipitation experiments, we confirmed the physical association of these proteins with the receptors. We then studied the functional relevance of the interaction between EPB41L2 and SREB1. Immunofluorescence microscopy revealed that SREB1 and EPB41L2 co-localize at the plasma membrane and that SREB1 is enriched in the ß-catenin-positive cell membranes. siRNA knockdown experiments revealed that EPB41L2 promotes the localization of SREB1 at the plasma membrane and increases the solubilization of SREB1 when using detergents, suggesting a modification of its membrane microenvironment. Altogether, these data suggest that EPB41L2 could regulate the subcellular compartmentalization of SREBs and, as proposed for other GPCRs, could affect their stability or activation.

REST-restrained lncRNA EPB41L4A-AS2 Modulates Laryngeal Squamous Cell Carcinoma Development via Regulating miR-1254/HIPK2 Pathway.

BACKGROUND: LncRNAs have been corroborated to exert crucial effects in malignancies, including laryngeal squamous cell carcinoma (LSCC). Nevertheless, the role and mechanism of EPB41L4A- AS2 in LSCC are inadequately investigated and warrant further exploration. METHODS: Relevant database was adopted to analyze the relationship between EPB41L4A-AS2 expression level and tumors. The expressions and relationships of EPB41L4A-AS2, RE-1 silencing transcription factor (REST), miR-1254, and homeodomain interacting protein kinase 2 (HIPK2) in LSCC cells were evaluated by qRT-PCR, Pearson's correlation tests, RNA immunoprecipitation, RNA pull-down assay, chromatin immunoprecipitation, database, and dual-luciferase reporter assay. Following the required transfection, the biological behaviors of LSCC cells were examined using cell function experiments. Meanwhile, the levels of Ki-67 and apoptosis-, and epithelial-mesenchymal transition (EMT) pathway-related proteins were quantified with Western blot. Moreover, xenografts in nude mice were constructed, and the tumor volume and weight were measured. Ki-67 positivity was determined by immunohistochemical staining. RESULTS: EPB41L4A-AS2 and HIPK2 were lower-expressed, yet miR-1254 and REST were higher- expressed in LSCC cells. Pearson's correlation assay results exhibited a positive correlation between HIPK2 and EPB41L4A-AS2 and a negative correlation between HIPK2 and miR-1254. Overexpressed EPB41L4A-AS2 diminished the biological behavior, and repressed the levels of Ki-67 and EMT-related markers in LSCC cells whilst enhancing those of apoptosis-related markers. These aforementioned effects were counteracted by miR-1254 mimic. Moreover, EPB41L4A- AS2 overexpression suppressed the growth of tumors and reduced the positive expression of Ki-67 in nude mice. Besides, miR-1254 aggravated the biological behaviors and elevated the levels of Ki-67 and EMT-related proteins in LSCC cells while reducing the levels of apoptosis-related markers via targeting HIPK2. CONCLUSION: REST-restrained EPB41L4A-AS2 modulates LSCC development via regulating miR-1254/HIPK2 pathway.

Expression pattern of lncRNAs in pituitary adenomas.

Non-functioning pituitary adenomas (NFPAs) are a group of pituitary tumors lacking manifestations linked to high hormone production, such as acromegaly and Cushing's syndrome. NFPA carcinogenesis depends on several molecular players. Long non-coding RNAs (lncRNAs) are a class of molecular players whose role in tumorigenesis has just recently been recognized. In the current study, we appraised expressions of 5 lncRNAs, namely FGD5-AS1, ATP6V0E2-AS1, ARHGAP5-AS1, WWC2-AS2 and EPB41L4A-AS1 in NFPAs versus their corresponding non-tumoral samples. Expressions of ATP6V0E2-AS1, EPB41L4A-AS1, FGD5-AS1 and WWC2-AS2 were significantly increased in NFPA samples compared with adjacent non-tumoral samples (P values = 0.037, 0.007, 0.008 and 0.03, respectively). However, expression of ARHGAP5-AS1 was not different between NFPA samples and controls (P value = 0.62). EPB41L4A-AS1 and FGD5-AS1 could discriminate between NFPA samples and adjacent non-tumoral samples (P values = 0.03 and 0.04, respectively). However, the AUC values were not appropriate. There was a significant positive association between age of NFPA patients and invasiveness of NFPA (χ2 = 4.24, P value = 0.039). Moreover, there was a significant positive association between diseases duration and CSF leak (χ2 = 11.4, p value = 0.023). Finally, there was a significant positive association between tumor size and Knosp classification (χ2 = 11.5, p value = 0.02) and invasiveness of NFPA (χ2 = 6.12, p value = 0.04). The current study provides information about dysregulation of lncRNAs in NFPAs and warrants additional studies in this field.

A YAP/TAZ-ARHGAP29-RhoA Signaling Axis Regulates Podocyte Protrusions and Integrin Adhesions.

Glomerular disease due to podocyte malfunction is a major factor in the pathogenesis of chronic kidney disease. Identification of podocyte-specific signaling pathways is therefore a prerequisite to characterizing relevant disease pathways and developing novel treatment approaches. Here, we employed loss of function studies for EPB41L5 (Yurt) as a central podocyte gene to generate a cell type-specific disease model. Loss of Yurt in fly nephrocytes caused protein uptake and slit diaphragm defects. Transcriptomic and proteomic analysis of human EPB41L5 knockout podocytes demonstrated impaired mechanotransduction via the YAP/TAZ signaling pathway. Further analysis of specific inhibition of the YAP/TAZ-TEAD transcription factor complex by TEADi led to the identification of ARGHAP29 as an EPB41L5 and YAP/TAZ-dependently expressed podocyte RhoGAP. Knockdown of ARHGAP29 caused increased RhoA activation, defective lamellipodia formation, and increased maturation of integrin adhesion complexes, explaining similar phenotypes caused by loss of EPB41L5 and TEADi expression in podocytes. Detection of increased levels of ARHGAP29 in early disease stages of human glomerular disease implies a novel negative feedback loop for mechanotransductive RhoA-YAP/TAZ signaling in podocyte physiology and disease.

The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis.

Novel genetic and epigenetic factors involved in the development and prognosis of idiopathic pulmonary fibrosis (IPF) have been identified. We previously observed that erythrocyte membrane protein band 4.1-like 3 (EPB41L3) increased in the lung fibroblasts of IPF patients. Thus, we investigated the role of EPB41L3 in IPF by comparing the EPB41L3 mRNA and protein expression of lung fibroblast between patients with IPF and controls. We also investigated the regulation of epithelial-mesenchymal transition (EMT) in an epithelial cell line (A549) and fibroblast-to-myofibroblast transition (FMT) in a fibroblast cell line (MRC5) by overexpressing and silencing EPB41L3. EPB41L3 mRNA and protein levels, as measured using RT-PCR, real-time PCR, and Western blot, were significantly higher in fibroblasts derived from 14 IPF patients than in those from 10 controls. The mRNA and protein expression of EPB41L3 was upregulated during transforming growth factor-ß-induced EMT and FMT. Overexpression of EPB41L3 in A549 cells using lenti-EPB41L3 transfection suppressed the mRNA and protein expression of N-cadherin and COL1A1. Treatment with EPB41L3 siRNA upregulated the mRNA and protein expression of N-cadherin. Overexpression of EPB41L3 in MRC5 cells using lenti-EPB41L3 transfection suppressed the mRNA and protein expression of fibronectin and α-SMA. Finally, treatment with EPB41L3 siRNA upregulated the mRNA and protein expression of FN1, COL1A1, and VIM. In conclusion, these data strongly support an inhibitory effect of EPB41L3 on the process of fibrosis and suggest the therapeutic potential of EPB41L3 as an anti-fibrotic mediator.

EPB41L4A-AS1 and UNC5B-AS1 have diagnostic and prognostic significance in osteosarcoma.

BACKGROUND: Deregulation of lncRNAs has been observed in human osteosarcoma. This study explored the diagnostic and prognostic significance of EPB41L4A-AS1 and UNC5B-AS1 in osteosarcoma. METHODS: Relative levels of EPB41L4A-AS1 and UNC5B-AS1 were detected in osteosarcoma tissue samples and cells. The ability to distinguish osteosarcoma from health was assessed by receiver operating characteristic (ROC) curve construction. Kaplan-Meier (K-M) and Cox proportional-hazards analyses were performed for prognosis factors. The bioinformatics approach was used to identify targeting miRNA for EPB41L4A-AS1 and UNC5B-AS1. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance. In cell culture experiments, the influence of EPB41L4A-AS1 and UNC5B-AS1 on proliferation, migration, and invasion of the osteosarcoma cell line was examined by CCK-8 and Transwell assays. RESULTS: Levels of EPB41L4A-AS1 and UNC5B-AS1 were upregulated in osteosarcoma patients and cells compared with the healthy participants and normal cell lines. EPB41L4A-AS1 and UNC5B-AS1 have a potent ability to distinguish the patients with osteosarcoma from the health. EPB41L4A-AS1 and UNC5B-AS1 levels correlated with SSS stage. Patients with high levels of EPB41L4A-AS1 and UNC5B-AS1 had significantly shorter survival times. EPB41L4A-AS1 and UNC5B-AS1 were independent prognostic indexes for overall survival. miR-1306-5p was a common target for EPB41L4A-AS1 and UNC5B-AS1. A propulsive impact on cell proliferation, migration, and invasion by EPB41L4A-AS1 and UNC5B-AS1 was observed, but can be rescued by miR-1306-5p. CONCLUSIONS: It was concluded that upregulations of EPB41L4A-AS1 and UNC5B-AS1 expression were diagnostic and prognostic biomarkers for human osteosarcoma. EPB41L4A-AS1 and UNC5B-AS1 contribute to the biological behavior of osteosarcoma via miR-1306-5p.

Five Years' Experience with Gene Panel Sequencing in Hereditary Hemolytic Anemia Screened by Routine Peripheral Blood Smear Examination.

BACKGROUND: Hereditary hemolytic anemia (HHA) is defined as a group of heterogeneous and rare diseases caused by defects of red blood cell (RBC) metabolism and RBC membrane, which leads to lysis or premature clearance. The aim of this study was to investigate individuals with HHA for potential disease-causing variants in 33 genes reported to be associated with HHA. METHODS: A total of 14 independent individuals or families diagnosed with suspected HHA, and in particular, RBC membranopathy, RBC enzymopathy, and hemoglobinopathy, were collected after routine peripheral blood smear testing. A custom designed panel, including the 33 genes, was performed using gene panel sequencing on the Ion Torrent PGM™ Dx System. The best candidate disease-causing variants were confirmed by Sanger sequencing. RESULTS: Several variants of the HHA-associated genes were detected in 10 out of 14 suspected HHA individuals. After excluding those variants predicted to be benign, 10 pathogenic variants and 1 variant of uncertain significance (VUS) were confirmed in 10 individuals with suspected HHA. Of these variants, the p.Trp704Ter nonsense variant of EPB41 and missense p.Gly151Asp variant of SPTA1 were identified in two out of four hereditary elliptocytoses. The frameshift p.Leu884GlyfsTer27 variant of ANK1, nonsense p.Trp652Ter variant of the SPTB, and missense p.Arg490Trp variant of PKLR were detected in all four hereditary spherocytosis cases. Missense p.Glu27Lys, nonsense p.Lys18Ter variants, and splicing errors such as c.92 + 1G > T and c.315 + 1G > A within HBB were identified in four beta thalassemia cases. CONCLUSIONS: This study provides a snapshot of the genetic alterations in a cohort of Korean HHA individuals and demonstrates the clinical utility of using gene panels in HHA. Genetic results can provide precise clinical diagnosis and guidance regarding medical treatment and management for some individuals.

[Novel BRAF::EPB41L2 gene fusion in posterior fossa pilocytic astrocytoma. Brief communication]. / Novyi tip sliyaniya genov BRAF::EPB41L2 v piloidnoi astrotsitome zadnei cherepnoi yamki. Kratkoe soobshchenie.

Identification of specific alterations in tumors (as a rule, these are mutations or gene fusions) makes it possible to prescribe targeted drugs of the second line of therapy or, in some cases of inoperable tumors, to observe not only a gradual partial response of the tumor to treatment, but also the removal of these patients from the category of incurable ones. The article describes a new rare type of BRAF::EPB41L2 gene fusion detected in a piloid astrocytoma that developed in the posterior cranial fossa in an 11-year-old boy.

Tumor Suppressor 4.1N/EPB41L1 is Epigenetic Silenced by Promoter Methylation and MiR-454-3p in NSCLC.

Non-small-cell lung cancer (NSCLC) is divided into three major histological types, namely, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and large-cell lung carcinoma (LCLC). We previously identified that 4.1N/EPB41L1 acts as a tumor suppressor and is reduced in NSCLC patients. In the current study, we explored the underlying epigenetic mechanisms of 4.1N/EPB41L1 reduction in NSCLC. The 4.1N/EPB41L1 gene promoter region was highly methylated in LUAD and LUSC patients. LUAD patients with higher methylation level in the 4.1N/EPB41L1 gene promoter (TSS1500, cg13399773 or TSS200, cg20993403) had a shorter overall survival time (Log-rank p = 0.02 HR = 1.509 or Log-rank p = 0.016 HR = 1.509), whereas LUSC patients with higher methylation level in the 4.1N/EPB41L1 gene promoter (TSS1500 cg13399773, TSS1500 cg07030373 or TSS200 cg20993403) had a longer overall survival time (Log-rank p = 0.045 HR = 0.5709, Log-rank p = 0.018 HR = 0.68 or Log-rank p = 0.014 HR = 0.639, respectively). High methylation of the 4.1N/EPB41L1 gene promoter appeared to be a relatively early event in LUAD and LUSC. DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine restored the 4.1N/EPB41L1 expression at both the mRNA and protein levels. MiR-454-3p was abnormally highly expressed in NSCLC and directly targeted 4.1N/EPB41L1 mRNA. MiR-454-3p expression was significantly correlated with 4.1N/EPB41L1 expression in NSCLC patients (r = -0.63, p < 0.0001). Therefore, we concluded that promoter hypermethylation of the 4.1N/EPB41L1 gene and abnormally high expressed miR-454-3p work at different regulation levels but in concert to restrict 4.1N/EPB41L1 expression in NSCLC. Taken together, this work contributes to elucidate the underlying epigenetic disruptions of 4.1N/EPB41L1 deficiency in NSCLC.

Circular RNA EPB41 expression predicts unfavorable prognoses in NSCLC by regulating miR-486-3p/eIF5A axis-mediated stemness.

Dysregulation of circular RNAs (circRNAs) has recently been found to play an important role in the progression and development of cancers such as non-small cell lung cancer (NSCLC). Yet the functions of many circRNAs in NSCLC remain unclear. In this study, the circRNA expression profiles in NSCLC tumor tissues and adjacent non-tumorous tissues were detected by high-throughput sequencing. Bioinformatics analyses, the dual-luciferase reporter system, fluorescence in situ hybridization (FISH) and miRNA/mRNA high-throughput sequencing were used to identify circ-EPB41 and its downstream target. The subcutaneous tumor/caudal vein transfer mouse model was used for tumor growth and invasion analysis. The results show that the circ-EPB41 was upregulated in NSCLC tissues and cell lines. Increased circ-EPB41 expression in NSCLC was significantly correlated with malignant characteristics, and positive to post-surgical overall survival of NSCLC patients. Reduced circ-EPB41 expression in NSCLC decreased cell proliferation and invasion in both in vitro and in vivo experiments. The miRNA/mRNA high-throughput sequencing suggested that downregulation of circ-EPB41 promoted microRNA (miR)-486-3p and suppressed eukaryotic translation initiation factor 5A (eIF5A) expression. Luciferase reporter experiments confirmed that miR-486-3p/eIF5A were downstream targets of circ-EPB41. In addition, we also found that downregulation of circ-EPB41 suppressed self-renewal and decreased expression of stemness markers SOX2, OCT-4, Nanog and CD133 by sponging miR-486-3p to enhance eIF5A expression. Taken togeter, these data revealed the important role of circ-EPB41 in regulating NSCLC cell invasion and proliferation by modifying miR-486-3p/eIF5A axis-mediated stemness. We believe our study provides a novel perspective regarding the role of circRNAs in NSCLC progression.

Persistent high glucose induced EPB41L4A-AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non-histones.

BACKGROUND: Persistent hyperglycemia decreases the sensitivity of insulin-sensitive organs to insulin, owing to which cells fail to take up and utilize glucose, which exacerbates the progression of type 2 diabetes mellitus (T2DM). lncRNAs' abnormal expression is reported to be associated with the progression of diabetes and plays a significant role in glucose metabolism. Herein, we study the detailed mechanism underlying the functions of lncRNA EPB41L4A-AS1in T2DM. METHODS: Data from GEO datasets were used to analyze the expression of EPB41L4A-AS1 between insulin resistance or type 2 diabetes patients and the healthy people. Gene expression was evaluated by qRT-PCR and western blotting. Glucose uptake was measured by Glucose Uptake Fluorometric Assay Kit. Glucose tolerance of mice was detected by Intraperitoneal glucose tolerance tests. Cell viability was assessed by CCK-8 assay. The interaction between EPB41L4A-AS1 and GCN5 was explored by RNA immunoprecipitation, RNA pull-down and RNA-FISH combined immunofluorescence. Oxygen consumption rate was tested by Seahorse XF Mito Stress Test. RESULTS: EPB41L4A-AS1 was abnormally increased in the liver of patients with T2DM and upregulated in the muscle cells of patients with insulin resistance and in T2DM cell models. The upregulation was associated with increased TP53 expression and reduced glucose uptake. Mechanistically, through interaction with GCN5, EPB41L4A-AS1 regulated histone H3K27 crotonylation in the GLUT4 promoter region and nonhistone PGC1ß acetylation, which inhibited GLUT4 transcription and suppressed glucose uptake by muscle cells. In contrast, EPB41L4A-AS1 binding to GCN5 enhanced H3K27 and H3K14 acetylation in the TXNIP promoter region, which activated transcription by promoting the recruitment of the transcriptional activator MLXIP. This enhanced GLUT4/2 endocytosis and further suppressed glucose uptake. CONCLUSION: Our study first showed that the EPB41L4A-AS1/GCN5 complex repressed glucose uptake via targeting GLUT4/2 and TXNIP by regulating histone and nonhistone acetylation or crotonylation. Since a weaker glucose uptake ability is one of the major clinical features of T2DM, the inhibition of EPB41L4A-AS1 expression seems to be a potentially effective strategy for drug development in T2DM treatment.

LncRNA EPB41L4A-AS1 Regulates Cell Proliferation, Apoptosis and Metastasis in Breast Cancer.

OBJECTIVE: Breast cancer is a leading global public health problem. In our previous study, we identified that lncRNA EPB41L4A antisense RNA 1 (EPB41L4A-AS1) was significantly downregulated in breast cancer. However, the functional role of EPB41L4A-AS1 in breast cancer has not been clarified. Here, we further confirmed the expression and biological function of EPB41L4A-AS1 in breast cancer. MATERIALS: To demonstrate the role of EPB41L4A-AS1 in breast cancer, we transfected breast cancer lines with pcDNA3.1-EPB41L4A-AS1 expression vector to induce ectopic overexpression of EPB41L4A-AS1. Then, to explore the role of EPB41L4A-AS1 overexpression in breast cancer cell growth, cell cycle, apoptosis, invasion, and migration capacity, we performed CCK-8 assay, colony formation assay, flow cytometry analysis, wound recovery and transwell assay, respectively. We also constructed a co-expression network to explore the potential effect mechanism of EPB41L4A-AS1. RESULTS: Our research showed EPB41L4A-AS1 expression was significantly lower in tumor tissues than in adjacent non-cancerous tissues. Overexpression of EPB41L4A-AS1 significantly reduced the proliferation of breast cancer cells. Flow cytometric analysis showed that forced expression of EPB41L4A-AS1 significantly increased the apoptosis rate of breast cancer cells. In addition, we found that upregulated EPB41L4A-AS1 significantly inhibited the migration and invasive ability of breast cancer. Functional analysis of co-expressed mRNAs suggested that EPB41L4A-AS1 may be involved in ribosomal, cell cycle, spliceosomal and p53 signaling pathways. CONCLUSION: Our findings suggest that EPB41L4A-AS1 is a tumor suppressor gene in breast cancer.

Down-regulation of EPB41L4A-AS1 mediated the brain aging and neurodegenerative diseases via damaging synthesis of NAD+ and ATP.

BACKGROUND: Aging and neurodegenerative diseases are typical metabolic-related processes. As a metabolism-related long non-coding RNA, EPB41L4A-AS has been reported to be potentially involved in the development of brain aging and neurodegenerative diseases. In this study, we sought to reveal the mechanisms of EPB41L4A-AS in aging and neurodegenerative diseases. METHODS: Human hippocampal gene expression profiles downloaded from the Genotype-Tissue Expression database were analyzed to obtain age-stratified differentially expressed genes; a weighted correlation network analysis algorithm was then used to construct a gene co-expression network of these differentially expressed genes to obtain gene clustering modules. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, protein-protein interaction network, and correlation analysis were used to reveal the role of EPB41L4A-AS1. The mechanism was verified using Gene Expression Omnibus dataset GSE5281 and biological experiments (construction of cell lines, Real-time quantitative PCR, Western blot, measurement of ATP and NAD+ levels, nicotinamide riboside treatment, Chromatin Immunoprecipitation) in neurons and glial-derived cells. RESULTS: EPB41L4A-AS1 was downregulated in aging and Alzheimer's disease. EPB41L4A-AS1 related genes were found to be enriched in the electron transport chain and NAD+ synthesis pathway. Furthermore, these genes were highly associated with neurodegenerative diseases and positively correlated with EPB41L4A-AS1. In addition, biological experiments proved that the downregulation of EPB41L4A-AS1 could reduce the expression of these genes via histone H3 lysine 27 acetylation, resulting in decreased NAD+ and ATP levels, while EPB41L4A-AS1 overexpression and nicotinamide riboside treatment could restore the NAD+ and ATP levels. CONCLUSIONS: Downregulation of EPB41L4A-AS1 not only disturbs NAD+ biosynthesis but also affects ATP synthesis. As a result, the high demand for NAD+ and ATP in the brain cannot be met, promoting the development of brain aging and neurodegenerative diseases. However, overexpression of EPB41L4A-AS1 and nicotinamide riboside, a substrate of NAD+ synthesis, can reduce EPB41L4A-AS1 downregulation-mediated decrease of NAD+ and ATP synthesis. Our results provide new perspectives on the mechanisms underlying brain aging and neurodegenerative diseases.

EPB41L5 promotes EMT through the ERK/p38 MAPK signaling pathway in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors worldwide and is characterized by activation of epithelial-mesenchymal transition (EMT). EPB41L5 is regarded as a key factor in the progression of EMT and metastasis in various kinds of cancers, although the role and mechanism of EPB41L5 in ESCC have not yet been elucidated. In addition, tumor cells can acquire enhanced aggressiveness and a mesenchymal phenotype through phosphorylation of MAPK signaling pathway components. Here, we intend to explore whether EPB41L5 can regulate the EMT process in ESCC and reveal whether the MAPK signaling pathway is involved. METHODS: We compared the expression level of EPB41L5 with the prognostic characteristics of 100 ESCC patients to hypothesize the role of EPB41L5 in the progression of ESCC. Furthermore, in vivo and in vitro experiments were conducted to verify the conclusions from the analysis of clinical specimens and investigate the underlying mechanism by which EPB41L5 contributes to ESCC. RESULTS: We discovered that EPB41L5 was overexpressed in ESCC and that higher EPB41L5 expression was related to higher TNM stage, a higher incidence of lymphatic metastasis and worse prognosis. Moreover, using ESCC cells and nude mouse models, we found that EPB41L5 promoted EMT, proliferation, migration and invasion in ESCC. Mechanistically, activation of phosphorylation in the ERK/p38 MAPK signaling pathway was involved in the EPB41L5-mediated regulation of EMT. CONCLUSION: In conclusion, our findings suggest that EPB41L5 plays a critical role in the regulation of EMT and the progression of ESCC.