LncRNAway: a web-based sgRNA design tool for precise and effective suppression of long noncoding RNAs.

Thousands of long noncoding RNAs (lncRNAs) have been annotated via high-throughput RNA sequencing, yet only a small fraction have been functionally investigated. Genomic knockout is the mainstream strategy for studying the biological function of protein-coding genes and lncRNAs, whereas the complexity of the lncRNA locus, especially the natural antisense lncRNAs (NAT-lncRNAs), presents great challenges. Knocking out lncRNAs often results in unintended disruptions of neighboring protein-coding genes and small RNAs, leading to ambiguity in observing phenotypes and interpreting biological function. To address this issue, we launched LncRNAway, a user-friendly web tool based on the BESST (branchpoint to 3' splicing site targeting) method, to design sgRNAs for lncRNA knockout. LncRNAway not only provides specific and effective lncRNA knockout guidelines but also integrates genotyping primers and quantitative PCR primers designing, thereby streamlining experimental procedures of lncRNA function study. LncRNAway is freely available at https://www.lncrnaway.com.

Anti-proliferation and apoptosis induced via the mTOR/PGC-1α signaling pathway in trophoblast cells of miscarriage.

Miscarriage is a common complication during early pregnancy and affects approximately 10%-15% of all pregnant women. Several studies have reported that the abnormal expression of mitochondrial oxidative stress-related genes might be involved in the occurrence and progression of miscarriage. The present study attempted to uncover the role of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in miscarriage chorionic villous tissue. The hypothesis that PGC-1α is crucial for glycolysis and oxidative phosphorylation during early pregnancy was tested. The results showed that the mRNA and protein levels of PGC-1α were significantly increased in the miscarriage chorionic villous tissues compared with the artificial selective abortion group, and that the expression was regulated by mTOR in knockdown and overexpression of mTOR in HTR8 cell lines. PGC-1α also promoted mitochondrion oxidative phosphorylation but inhibited glycolysis process. In addition, PGC-1α could drive ROS production, reduce mitochondrial membrane potential and block NADPH synthesis, resulting in cell cycle arrest and cell apoptosis, eventually leading to miscarriage. These results suggested that the aberrant expression of PGC-1α is involved in the etiology of early miscarriage, providing new perspectives regarding the mechanisms of miscarriage and a potential therapeutic target for miscarriage.

Mechanism Analysis of Discharge Energy in the Electrostatic-Field-Induced Electrolyte Jet Micro-EDM.

The discharge energy determines the machining resolution, minimum processable feature size, and surface roughness, which makes it a hot research topic in the microelectrical discharge machining (EDM) field. In this paper, a kind of novel discharge-energy-generation method in micro-EDM is investigated. In this method, the opposite induced charges on the electrolyte jet and workpiece serve as the source of the discharge energy. The operating mechanism of this discharge energy is revealed by analyzing the equivalent discharge circuit. The unique discharge current and voltage between the electrolyte jet and the workpiece are sampled and investigated. In contrast with the pulsating energy in conventional EDM, this study shows that the direct current (DC) voltage source can automatically generate a continuously periodical pulsating discharge in the electrostatic-field-induced electrolyte jet (E-Jet) EDM process. After further analyzing the electric signals in a single discharge process, it can be found that the interelectrode voltage experienced a continuous sharp electric breakdown, a nearly unchanging process, and a fast exponential recharging process. The discharge frequency increases as the electrolyte concentration and interelectrode voltage increase but decreases as the interelectrode distance increases. The discharge energy per pulse increases with the increasing interelectrode distance and electrolyte concentration but with the decreasing interelectrode voltage. Finally, the electrostatic-field-induced discharge-energy generation and change mechanisms are revealed, which provides a feasible method for micro-EDM with continuous tiny pulsed energy only using the DC power supply.

Laser Processing and Multi-Energy Field Manufacturing of High-Performance Materials.

The laser is one of the major inventions of the 20th century, along with atomic energy, the computer and semiconductors [...].

Antibacterial Activity and Mechanism of Canagliflozin against Methicillin-Resistant Staphylococcus aureus.

Diabetic foot infection (DFI) is a common complication in diabetes patients, with foot infections being the leading cause of amputations. Staphylococcus aureus is frequently found in diabetic foot infections, of which methicillin-resistant Staphylococcus aureus (MRSA) has become a major clinical and epidemiological challenge. Since MRSA strains are resistant to most ß-lactam antibiotics, and also partially resistant to other antibiotics, treatment is difficult and costly. The emergence of drug-resistant bacteria often arises from overuse or misuse of antibiotics. Clinically, canagliflozin is commonly used for the treatment of type 2 diabetes. On this basis, we investigated the antibacterial activity and mechanism of canagliflozin against MRSA, with the aim to discover novel functions of canagliflozin and provide new insights for the treatment of MRSA. Using the microbroth dilution method to determine the half maximal inhibitory concentration of drugs, we found that canagliflozin not only can inhibit the growth of methicillin-sensitive Staphylococcus aureus (MSSA) but also exhibits antibacterial activity against MRSA. The IC50 values, at approximately 56.01 µM and 57.60 µM, were almost the same. At 12 h, canagliflozin showed a significant antibacterial effect against MRSA at and above 30 µM. In addition, its combined use with penicillin achieved better antibacterial effects, which were increased by about three times. Additive antibacterial activity (FICI = 0.69) was found between penicillin and canagliflozin, which was better than that of doxycycline and canagliflozin (FICI = 0.95). Canagliflozin also affected bacterial metabolic markers, such as glucose, ATP, and lactic acid. The results of crystal violet staining indicate that canagliflozin disrupted the formation of bacterial biofilm. Our electron microscopy results showed that canagliflozin distorted the bacterial cell wall. The results of RT-PCR suggest that canagliflozin down-regulated the expressions of biofilm-related gene (clfA, cna, agrC, mgrA, hld) and methicillin-resistance gene (mecA), which was related to MRSA. Molecular docking also indicated that canagliflozin affected some interesting targets of MRSA, such as the sarA, crtM and fnbA proteins. In conclusion, canagliflozin exhibits antibacterial activity against MRSA by affecting bacterial metabolism, inhibiting its biofilm formation, distorting the bacterial cell wall, and altering the gene expression of biofilm formation and its virulence. Our study reveals the antibacterial activity of canagliflozin against MRSA, providing a new reference for treating diabetic foot infections.

Research on Electrostatic Field-Induced Discharge Energy in Conventional Micro EDM.

The electrostatic field-induced electrolyte jet (E-Jet) electric discharge machining (EDM) is a newly developed micro machining method. However, the strong coupling of the electrolyte jet liquid electrode and the electrostatic induced energy prohibited it from utilization in conventional EDM process. In this study, the method with two discharge devices connecting in serials is proposed to decouple pulse energy from the E-Jet EDM process. By automatic breakdown between the E-Jet tip and the auxiliary electrode in the first device, the pulsed discharge between the solid electrode and the solid workpiece in the second device can be generated. With this method, the induced charges on the E-Jet tip can indirectly regulate the discharge between the solid electrodes, giving a new pulse discharge energy generation method for traditional micro EDM. The pulsed variation of current and voltage generated during the discharge process in conventional EDM process verified the feasibility of this decoupling approach. The influence of the distance between the jet tip and the electrode, as well as the gap between the solid electrode and the work-piece, on the pulsed energy, demonstrates that the gap servo control method is applicable. Experiments with single points and grooves indicate the machining ability of this new energy generation method.

SQSTM1/p62 Knockout by Using the CRISPR/Cas9 System Inhibits Migration and Invasion of Hepatocellular Carcinoma.

Migration and invasion play crucial roles in the progression of hepatocellular carcinoma (HCC), but the underlying mechanisms are not clear. Analysis of clinical samples indicates that SQSTM1/p62 is highly expressed in HCC and seriously affects the prognosis of patients. Subsequently, we showed that SQSTM1/p62 knockout using the CRISPR/Cas9 system led to impaired migration and invasion of HCC, upregulated Keap1, and promoted the inhibitory effect of Keap1 on Nrf2. Then, the inactivation of Nrf2 inhibited the expression of matrix metalloproteinases (MMPs), thus attenuating the migration and invasion of HCC. We also found that SQSTM1/p62 knockout significantly inhibited migration and invasion in a lung metastasis model of nude mice with HCC. Furthermore, we found that cisplatin not only significantly inhibited the expression of SQSTM1/p62 but also slowed down the migration and invasion of HCC, while the inflammatory microenvironment accelerated the migration and invasion of HCC. These results suggest for the first time that SQSTM1/p62 knockout inhibits the migration and invasion of HCC through the Keap1/Nrf2/MMP2 signaling pathway. SQSTM1/p62 may be developed into a key drug target to regulate the migration and invasion of HCC cells.

Canagliflozin Delays Aging of HUVECs Induced by Palmitic Acid via the ROS/p38/JNK Pathway.

Vascular aging is an important factor contributing to cardiovascular diseases, such as hypertension and atherosclerosis. Hyperlipidemia or fatty accumulation may play an important role in vascular aging and cardiovascular diseases. Canagliflozin (CAN), a sodium-glucose cotransporter inhibitor, can exert a cardiovascular protection effect that is likely independent of its hypoglycemic activities; however, the exact mechanisms remain undetermined. We hypothesized that CAN might have protective effects on blood vessels by regulating vascular aging induced by hyperlipidemia or fatty accumulation in blood vessel walls. In this study, which was undertaken on the basis of aging and inflammation, we investigated the protective effects and mechanisms of CAN in human umbilical vein endothelial cells induced by palmitic acid. We found that CAN could delay vascular aging, reduce the secretion of the senescence-associated secretory phenotype (SASP) and protect DNA from damage, as well as exerting an effect on the cell cycle of senescent cells. These actions likely occur through the attenuation of the excess reactive oxygen species (ROS) produced in vascular endothelial cells and/or down-regulation of the p38/JNK signaling pathway. In summary, our study revealed a new role for CAN as one of the sodium-dependent glucose transporter 2 inhibitors in delaying lipotoxicity-induced vascular aging by targeting the ROS/p38/JNK pathway, giving new medicinal value to CAN and providing novel therapeutic ideas for delaying vascular aging in patients with dyslipidemia.

BESST: a novel LncRNA knockout strategy with less genome perturbance.

Long noncoding RNAs (lncRNAs) are >200 nt RNA transcripts without protein-coding potential. LncRNAs can be categorized into intergenic, intronic, bidirectional, sense, and antisense lncRNAs based on the genomic localization to nearby protein-coding genes. The current CRISPR-based lncRNA knockout strategy works efficiently for lncRNAs distant from the protein-coding gene, whereas it causes genomic perturbance inevitably due to technical limitations. In this study, we introduce a novel lncRNA knockout strategy, BESST, by deleting the genomic DNA fragment from the branch point to the 3' splicing site in the last intron of the target lncRNA. The BESST knockout exhibited comparable or superior repressive efficiency to RNA silencing or conventional promoter-exon1 deletion. Significantly, the BESST knockout strategy minimized the intervention of adjacent/overlap protein-coding genes by removing an average of ∼130 bp from genomic DNA. Our data also found that the BESST knockout strategy causes lncRNA nuclear retention, resulting in decapping and deadenylation of the lncRNA poly(A) tail. Further study revealed that PABPN1 is essential for the BESST-mediated decay and subsequent poly(A) deadenylation and decapping. Together, the BESST knockout strategy provides a versatile tool for investigating gene function by generating knockout cells or animals with high specificity and efficiency.

Canagliflozin Attenuates Lipotoxicity in Cardiomyocytes by Inhibiting Inflammation and Ferroptosis through Activating AMPK Pathway.

Diabetic cardiomyopathy (DCM) is a myocardial disease independent of other cardiovascular diseases, such as coronary heart disease, hypertension, etc. Lipotoxicity is closely related to DCM. In this study, we investigated the mechanism of lipid metabolism disturbance in DCM in HL-1 cells. Through bioinformatics and Western blotting analysis, we found that canagliflozin (CAN) significantly inhibited the expression of inflammatory factors cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Ferroptosis is mediated by lipid peroxidation. We demonstrated the presence of ferroptosis in cardiomyocytes by detecting intracellular Fe2+ content and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), and mitochondrial membrane potential (MMP). CAN could significantly regulate the indicators of ferroptosis. By using specific inhibitors celecoxib (coxib), S-methylisothiourea sulfate (SMT), Ferrostatin-1 (Fer-1), and Compound C, we further found that CAN regulated inflammation and ferroptosis through AMP-activated protein (AMPK), and inflammation interacted with ferroptosis. Our study indicated that CAN attenuated lipotoxicity in cardiomyocytes by regulating inflammation and ferroptosis through activating the AMPK pathway. This study provides a new direction of myocardial lipotoxicity and some new information for the treatment of DCM.

Autophagy markers are dysregulated in the endometrial tissues of patients with unexplained repeated implantation failure.

Endometrium decidualization is a complex biological process, which includes the interplay of transcription factors, cytokines, cell cycle regulators, and other signaling pathways. However, the underlying molecular mechanisms of this process are not fully elucidated to date. In this study, we aimed to investigate the possible association between autophagy and recurrent implantation failure (RIF). A total of 81 genes were downregulated and 231 genes were upregulated in the RIF group compared with the control group, and the differences were statistically significant (p < 0.05). Further, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway were analyzed, and we found that some autophagy markers, for example, LC3-II, LAMP2, and HIF-1α were significantly increased, whereas P62 was drastically downregulated in the RIF group. Similar results were observed in proteins level; and the autophagy puncta were also markedly enhanced in the endometrial tissues of RIF patients. Autophagy is closely associated with the RIF occurs and may be involved in the pathogenesis of RIF.

LINC00472 inhibits cell migration by enhancing intercellular adhesion and regulates H3K27ac level via interacting with P300 in renal clear cell carcinoma.

Renal clear cell carcinoma (RCCC) is the most common type of renal cell carcinoma, which is also difficult to diagnose and easy to metastasize. Currently, there is still a lack of effective clinical diagnostic indicators and treatment targets. This study aims to find effective diagnostic markers and therapeutic targets from the perspective of noncoding RNA. In this study, we found that the expression of Long noncoding RNA LINC00472 was significantly decreased in RCCC and showed a downward trend with the progression of cancer stage. Patients with low LINC00472 expression have poor prognosis. Inhibition of LINC00472 significantly increased cell proliferation and migration, while overexpression of LINC00472 obviously inhibited cell proliferation and enhanced intercellular adhesion. Transcriptome sequencing analysis demonstrated that LINC00472 was highly correlated with extracellular matrix and cell metastasis-related pathways, and the consistent results were obtained by The Cancer Genome Atlas (TCGA) data analysis. Additionally, we discovered that the integrin family protein ITGB8 is a potential target gene of LINC00472. Mechanistically, we found that the change of LINC00472 affected the acetylation level of H3K27 site in cells, and we speculate that this effect is likely to be generated through the interaction with acetyltransferase P300. In conclusion, LINC00472 has an important impact on the proliferation and metastasis of renal clear cells, and probably participate in the regulation of histone modification, and it may be used as a potential diagnostic marker of RCCC.

A Whole Exon Screening-Based Score Model Predicts Prognosis and Immune Checkpoint Inhibitor Therapy Effects in Low-Grade Glioma.

Objective: This study aims to identify prognostic factors for low-grade glioma (LGG) via different machine learning methods in the whole genome and to predict patient prognoses based on these factors. We verified the results through in vitro experiments to further screen new potential therapeutic targets. Method: A total of 940 glioma patients from The Cancer Genome Atlas (TCGA) and The Chinese Glioma Genome Atlas (CGGA) were included in this study. Two different feature extraction algorithms - LASSO and Random Forest (RF) - were used to jointly screen genes significantly related to the prognosis of patients. The risk signature was constructed based on these screening genes, and the K-M curve and ROC curve evaluated it. Furthermore, we discussed the differences between the high- and low-risk groups distinguished by the signature in detail, including differential gene expression (DEG), single-nucleotide polymorphism (SNP), copy number variation (CNV), immune infiltration, and immune checkpoint. Finally, we identified the function of a novel molecule, METTL7B, which was highly correlated with PD-L1 expression on tumor cell, as verified by in vitro experiments. Results: We constructed an accurate prediction model based on seven genes (AUC at 1, 3, 5 years= 0.91, 0.85, 0.74). Further analysis showed that extracellular matrix remodeling and cytokine and chemokine release were activated in the high-risk group. The proportion of multiple immune cell infiltration was upregulated, especially macrophages, accompanied by the high expression of most immune checkpoints. According to the in vitro experiment, we preliminarily speculate that METTL7B affects the stability of PD-L1 mRNA by participating in the modification of m6A. Conclusion: The seven gene signatures we constructed can predict the prognosis of patients and identify the potential benefits of immune checkpoint inhibitors (ICI) therapy for LGG. More importantly, METTL7B, one of the risk genes, is a crucial molecule that regulates PD-L1 and could be used as a new potential therapeutic target.

Empagliflozin-Enhanced Antioxidant Defense Attenuates Lipotoxicity and Protects Hepatocytes by Promoting FoxO3a- and Nrf2-Mediated Nuclear Translocation via the CAMKK2/AMPK Pathway.

Lipotoxicity is an important factor in the development and progression of nonalcoholic steatohepatitis. Excessive accumulation of saturated fatty acids can increase the substrates of the mitochondrial electron transport chain in hepatocytes and cause the generation of reactive oxygen species, resulting in oxidative stress, mitochondrial dysfunction, loss of mitochondrial membrane potential, impaired triphosphate (ATP) production, and fracture and fragmentation of mitochondria, which ultimately leads to hepatocellular inflammatory injuries, apoptosis, and necrosis. In this study, we systematically investigated the effects and molecular mechanisms of empagliflozin on lipotoxicity in palmitic acid-treated LO2 cell lines. We found that empagliflozin protected hepatocytes and inhibited palmitic acid-induced lipotoxicity by reducing oxidative stress, improving mitochondrial functions, and attenuating apoptosis and inflammation responses. The mechanistic study indicated that empagliflozin significantly activated adenosine 5'-monophosphate (AMP)-activated protein kinase alpha (AMPKα) through Calcium/Calmodulin dependent protein kinase kinase beta (CAMKK2) instead of liver kinase B1 (LKB1) or TGF-beta activated kinase (TAK1). The activation of empagliflozin on AMPKα not only promoted FoxO3a phosphorylation and thus forkhead box O 3a (FoxO3a) nuclear translocation, but also promoted Nrf2 nuclear translocation. Furthermore, empagliflozin significantly upregulated the expressions of antioxidant enzymes superoxide dismutase (SOD) and HO-1. In addition, empagliflozin did not attenuate lipid accumulation at all. These results indicated that empagliflozin mitigated lipotoxicity in saturated fatty acid-induced hepatocytes, likely by promoting antioxidant defense instead of attenuating lipid accumulation through enhanced FoxO3a and Nrf2 nuclear translocation dependent on the CAMKK2/AMPKα pathway. The CAMKK2/AMPKα pathway might serve as a promising target in treatment of lipotoxicity in nonalcoholic steatohepatitis.

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.

Canagliflozin Modulates Hypoxia-Induced Metastasis, Angiogenesis and Glycolysis by Decreasing HIF-1α Protein Synthesis via AKT/mTOR Pathway.

The microenvironment plays a vital role in tumor progression, and hypoxia is a typical microenvironment feature in nearly all solid tumors. In this study, we focused on elucidating the effect of canagliflozin (CANA), a new class of antidiabetic agents, on hepatocarcinoma (HCC) tumorigenesis under hypoxia, and demonstrated that CANA could significantly inhibit hypoxia-induced metastasis, angiogenesis, and metabolic reprogramming in HCC. At the molecular level, this was accompanied by a reduction in VEGF expression level, as well as a reduction in the epithelial-to-mesenchymal transition (EMT)-related proteins and glycolysis-related proteins. Next, we focused our study particularly on the modulation of HIF-1α by CANA, which revealed that CANA decreased HIF-1α protein level by inhibiting its synthesis without affecting its proteasomal degradation. Furthermore, the AKT/mTOR pathway, which plays an important role in HIF-1α transcription and translation, was also inhibited by CANA. Thus, it can be concluded that CANA decreased metastasis, angiogenesis, and metabolic reprogramming in HCC by inhibiting HIF-1α protein accumulation, probably by targeting the AKT/mTOR pathway. Based on our results, we propose that CANA should be evaluated as a new treatment modality for liver cancer.

A Class of Protein-Coding RNAs Binds to Polycomb Repressive Complex 2 and Alters Histone Methylation.

Polycomb repressive complex 2 (PRC2) is a multi-subunit protein complex mediating the methylation of lysine 27 on histone H3 and playing an important role in transcriptional repression during tumorigenesis and development. Previous studies revealed that both protein-coding and non-coding RNAs could bind to PRC2 complex. However, the functions of protein-coding RNAs that bind to PRC2 complex in tumor are still unknown. Through data mining and RNA immunoprecipitation (RIP) assay, our study found that there were a class of protein-coding RNAs bound to PRC2 complex and H3 with tri-methylation on lysine 27. The Bayesian gene regulatory network analysis pointed out that these RNAs regulated the expression of PRC2-regulated genes in cancer. In addition, gene set enrichment analysis (GSEA), gene ontology (GO) analysis, and weighted gene co-expression network analysis (WGCNA) also confirmed that these RNAs were associated with histone modification in cancer. We also confirmed that MYO1C, a PRC2-bound transcript, inhibited the modification level of H3K27me3. Further detailed study showed that TMEM117 regulated TSLP expression through EZH2-mediated H3K27me3 modification. Interestingly, the RNA recognition motif of PRC2 complex might help these RNAs bind to the PRC2 complex more easily. The same regulatory pattern was found in mice as well.

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.

RNA-binding Protein MBNL2 regulates Cancer Cell Metastasis through MiR-182-MBNL2-AKT Pathway.

The aberrant expression of RNA-binding proteins (RBPs) plays important roles in the occurrence and progression of cancer. MBNL2 is a member of the RNA binding protein MBNL family that is widely expressed in mammalian cells. We report here that MBNL2 is downregulated in breast, lung and liver cancer tissues, the promoter methylation levels of MBNL2 are higher in cancer tissues than normal tissues. The enrichment analysis of MBNL2 correlated genes indicates the potential function of MBNL2 on cancer progression. MBNL2 regulates cancer cell migration and invasion by modulating PI3K/AKT-mediated epithelial-mesenchymal transition. PI3K/AKT inhibitor overcomes the promotive effect of shMBNL2 on metastasis. The expression of MBNL2 is directly targeted by miR-182. miR-182 is upregulated in breast, lung and liver cancers and has good potential for cancer diagnosis. miR-182 promotes cancer cell migration and invasion by inhibiting the expression of MBNL2. Re-introduction of exogenous MBNL2 reverses the promotive effect of miR-182 on metastasis. Collectively, these findings suggest that MBNL2 plays a tumor suppressive function through miR-182-MBNL2-AKT-EMT signaling pathways.