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.

Functional knockout of long non-coding RNAs with genome editing.

An effective loss-of-function study is necessary to investigate the biological function of long non-coding RNA (lncRNA). Various approaches are available, including RNA silencing, antisense oligos, and CRISPR-based genome editing. CRISPR-based genome editing is the most widely used for inactivating lncRNA function at the genomic level. Knocking out the lncRNA function can be achieved by removing the promoter and the first exon (PE1), introducing pre-termination poly(A) signals, or deleting the entire locus, unlike frameshift strategies used for messenger RNA (mRNA). However, the intricate genomic interplay between lncRNA and neighbor genes makes it challenging to interpret lncRNA function accurately. This article discusses the advantages and disadvantages of each lncRNA knockout method and envisions the potential future directions to facilitate lncRNA functional study.

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.

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.

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 NEAT1 induces autophagy through epigenetic regulation of autophagy-related gene expression in neuroglial cells.

Endocytosis and autophagy are two important pathways for amyloid-ß (Aß) clearance in neuroglial cells. Our previous study demonstrated that nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding RNA modulates Aß clearance mediated by neuroglial cells via the epigenetic regulation of endocytosis-related genes. Herein, we demonstrate that NEAT1 functions as an autophagy inducer by modulating the expression of multiple autophagy-related genes, including autophagy-related 5 (atg5), autophagy-related 3 (atg3), and beclin1. NEAT1 can promote transcription of these genes by altering histone modification near these transcriptional start sites of the genes and thereby influencing the recruitment of signal transducer and activator of transcription 3 to these gene promoters. Our findings demonstrate a new cellular function of NEAT1 in neuroglial cells and suggest a potential therapeutic target for the treatment of autophagy-related diseases.

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.

Upregulation of the APOBEC3 Family Is Associated with a Poor Prognosis and Influences Treatment Response to Raf Inhibitors in Low Grade Glioma.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) has been identified as a group of enzymes that catalyze cytosine deamination in single-stranded (ss) DNA to form uracil, causing somatic mutations in some cancers. We analyzed the APOBEC3 family in 33 TCGA cancer types and the results indicated that APOBEC3s are upregulated in multiple cancers and strongly correlate with prognosis, particularly in low grade glioma (LGG). Then we constructed a prognostic model based on family expression in LGG where the APOBEC3 family signature is an accurate predictive model (AUC of 0.85). Gene mutation, copy number variation (CNV), and a differential gene expression (DEG) analysis were performed in different risk groups, and the weighted gene co-expression network analysis (WGCNA) was employed to clarify the role of various members in LGG; CIBERSORT algorithm was deployed to evaluate the landscape of LGG immune infiltration. We found that upregulation of the APOBEC3 family expression can strengthen Ras/MAPK signaling pathway, promote tumor progression, and ultimately reduce the treatment benefits of Raf inhibitors. Moreover, the APOBEC3 family was shown to enhance the immune response mediated by myeloid cells and interferon gamma, as well as PD-L1 and PD-L2 expression, implying that they have immunotherapy potential. Therefore, the APOBEC3 signature enables an efficient assessment of LGG patient survival outcomes and expansion of clinical benefits by selecting appropriate individualized treatment strategies.

NEAT1 regulates microtubule stabilization via FZD3/GSK3ß/P-tau pathway in SH-SY5Y cells and APP/PS1 mice.

Nuclear paraspeckles assembly transcript 1 (NEAT1) is a well-known long noncoding RNA (lncRNA) with various functions in different physiological and pathological processes. Notably, aberrant NEAT1 expression is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular mechanism of NEAT1 in AD remains poorly understood. In this study, we investigated that NEAT1 regulated microtubules (MTs) polymerization via FZD3/GSK3ß/p-tau pathway. Downregulation of NEAT1 inhibited Frizzled Class Receptor 3 (FZD3) transcription activity by suppressing H3K27 acetylation (H3K27Ac) at the FZD3 promoter. Our data also demonstrated that P300, an important histone acetyltransferases (HAT), recruited by NEAT1 to bind to FZD3 promoter and mediated its transcription via regulating histone acetylation. In addition, according to immunofluorescence staining of MTs, metformin, a medicine for the treatment of diabetes mellitus, rescued the reduced length of neurites detected in NEAT1 silencing cells. We suspected that metformin may play a neuroprotective role in early AD by increasing NEAT1 expression and through FZD3/GSK3ß/p-tau pathway. Collectively, NEAT1 regulates microtubule stabilization via FZD3/GSK3ß/P-tau pathway and influences FZD3 transcription activity in the epigenetic way.

Overexpression of lncRNA EPB41L4A-AS1 Induces Metabolic Reprogramming in Trophoblast Cells and Placenta Tissue of Miscarriage.

Long non-coding RNAs (lncRNAs) have been shown to be crucial regulators in numerous human diseases. However, little is known about their effects on early recurrent miscarriage (RM). Here we aimed to investigate the role of lncRNA EPB41L4A-AS1 on placental trophoblast cell metabolic reprogramming, which might be involved in the pathogenesis of RM. After microarray and GEO database analyses, we found that EPB41L4A-AS1 was significantly increased in early RM placental tissue, and this increase may relate to estradiol-mediated upregulation of PGC-1α. EPB41L4A-AS1 overexpression inhibits glycolysis but increases the dependence on fatty acid oxidation in mitochondrion metabolism and suppresses the Warburg effect, which is necessary for rapid growth of the placental villus, leading to miscarriage. Mechanistic analyses demonstrated that EPB41L4A-AS1 functions as a lncRNA in the regulation of VDAC1 and HIF-1α expression through enhancement of H3K4me3 levels in the promoters of VDAC1 and HIF1A-AS1, a natural antisense transcript (NAT) lncRNA of HIF-1α. Taken together, these findings demonstrate that aberrant expression of EPB41L4A-AS1 is involved in the etiology of early RM, and it may be a candidate diagnostic hallmark and a potential therapeutic target for early RM treatment.

NEAT1 regulates neuroglial cell mediating Aß clearance via the epigenetic regulation of endocytosis-related genes expression.

The accumulation of intracellular ß-amyloid peptide (Aß) is important pathological characteristic of Alzheimer's disease (AD). However, the exact underlying molecular mechanism remains to be elucidated. Here, we reported that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a long n on-coding RNA, exhibits repressed expression in the early stage of AD and its down-regulation declines neuroglial cell mediating Aß clearance via inhibiting expression of endocytosis-related genes. We find that NEAT1 is associated with P300/CBP complex and its inhibition affects H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cro) located nearby to the transcription start site of many genes, including endocytosis-related genes. Interestingly, NEAT1 inhibition down-regulates H3K27Ac but up-regulates H3K27Cro through repression of acetyl-CoA generation. NEAT1 also mediates the binding between STAT3 and H3K27Ac but not H3K27Cro. Therefore, the decrease of H3K27Ac and/or the increase of H3K27Cro declines expression of multiple related genes. Collectively, this study first reveals the different roles of H3K27Ac and H3K27Cro in regulation of gene expression and provides the insight of the epigenetic regulatory mechanism of NEAT1 in gene expression and AD pathology.

LncRNA EPB41L4A-AS1 regulates glycolysis and glutaminolysis by mediating nucleolar translocation of HDAC2.

BACKGROUND: LncRNAs have been found to be involved in various aspects of biological processes. In this study, we aimed to uncover the molecular mechanisms of lncRNA EPB41L4A-AS1 in regulating glycolysis and glutaminolysis in cancer cells. METHODS: The expression of EPB41L4A-AS1 in cancer patients was analyzed in TCGA and GEO datasets. The level of cellular metabolism was determined by extracellular flux analyzer. The relationship between p53 and EPB41L4A-AS1 was explored by qRT-PCR, luciferase assay and ChIP assay. The interactions between EPB41L4A-AS1 and HDAC2 or NPM1 were determined by RNA immunoprecipitation, RNA pull-down assay and RNA-FISH- immunofluorescence. FINDINGS: EPB41L4A-AS1 was a p53-regulated gene. Low expression and deletion of lncRNA EPB41L4A-AS1 were found in a variety of human cancers and associated with poor prognosis of cancer patients. Knock down EPB41L4A-AS1 expression triggered Warburg effect, demonstrated as increased aerobic glycolysis and glutaminolysis. EPB41L4A-AS1 interacted and colocalized with HDAC2 and NPM1 in nucleolus. Silencing EPB41L4A-AS1 reduced the interaction between HDAC2 and NPM1, released HDAC2 from nucleolus and increased its distribution in nucleoplasm, enhanced HDAC2 occupation on VHL and VDAC1 promoter regions, and finally accelerated glycolysis and glutaminolysis. Depletion of EPB41L4A-AS1 increased the sensitivity of tumor to glutaminase inhibitor in tumor therapy. INTERPRETATION: EPB41L4A-AS1 functions as a repressor of the Warburg effect and plays important roles in metabolic reprogramming of cancer.

Relationship between LINC00341 expression and cancer prognosis.

LINC00341 is a novel long intergenic non-protein coding RNA with unknown functions. In our report, we investigated LINC00341 expression and its prognostic value in cancer patients. DNA over-methylation triggered low expression of LINC00341 and that was associated with poor prognosis in cancers. A meta-analysis further confirmed that high expression of LINC00341 was associated with a better prognosis in cancer patients. Both gene set enrichment analysis and meta-analysis showed that LINC00341 inhibited cancer metastasis. Finally, a large-scale multicentre analysis supported a prognostic value of LINC00341 in cancers.

NEAT1 modulates herpes simplex virus-1 replication by regulating viral gene transcription.

Nuclear paraspeckle assembly transcript 1 (NEAT1) is the crucial structural platform of paraspeckles, which is one type of nuclear bodies. As a stress-induced lncRNA, the expression of NEAT1 increases in response to viral infection, but little is known about the role of NEAT1 or paraspeckles in the replication of herpes simplex virus-1 (HSV-1). Here, we demonstrate that HSV-1 infection increases NEAT1 expression and paraspeckle formation in a STAT3-dependent manner. NEAT1 and other paraspeckle protein components, P54nrb and PSPC1, can associate with HSV-1 genomic DNA. By binding with STAT3, PSPC1 is required for the recruitment of STAT3 to paraspeckles and facilitates the interaction between STAT3 and viral gene promoters, finally increasing viral gene expression and viral replication. Furthermore, thermosensitive gel containing NEAT1 siRNA or STAT3 siRNA effectively healed the skin lesions caused by HSV-1 infection in mice. Our results provide insight into the roles of lncRNAs in the epigenetic control of viral genes and into the function of paraspeckles.

Assessment of a novel recombinant vesicular stomatitis virus with triple mutations in its matrix protein as a vaccine for pigs.

Vesicular stomatitis virus (VSV) causes a serious vesicular disease responsible for economic losses in the livestock industry. Currently, there are no suitable vaccines to prevent VSV infection. Although the structural matrix (M) protein of VSV has been shown to be a virulence factor in rodent models, its role in the pathogenicity of VSV infection in livestock species is unknown. We hypothesized that VSV with mutations in the M protein represents a novel live attenuated vaccine candidate. To test this, we introduced mutations into VSV M protein using reverse genetics and assessed their attenuation both in vitro and in pigs, an important natural host of VSV. A recombinant VSV with a triple amino acid mutation in M protein (VSVMT) demonstrated a significantly reduced ability to inhibit the type I interferon (IFN) signaling pathway and to shutoff host gene expression compared to WT-VSV and a mutant virus with a single amino acid deletion (VSVΔM51). Inoculation of pigs with VSVMT induced no apparent vesicular lesions but stimulated virus-neutralizing antibodies and animals were protected against virulent VSV challenge infection. These data demonstrate that the M protein is an important virulence factor for VSV in swine and VSVMT represents a novel vaccine candidate for VSV infections in pigs.

Evaluation of attenuated VSVs with mutated M or/and G proteins as vaccine vectors.

Vesicular stomatitis virus (VSV) is a promising vector for vaccine and oncolysis, but it can also produce acute diseases in cattle, horses, and swine characterized by vesiculation and ulceration of the tongue, oral tissues, feet, and teats. In experimental animals (primates, rats, and mice), VSV has been shown to lead to neurotoxicities, such as hind limb paralysis. The virus matrix protein (M) and glycoprotein (G) are both major pathogenic determinants of wild-type VSV and have been the major targets for the production of attenuated strains. Existing strategies for attenuation included: (1) deletion or M51R substitution in the M protein (VSVΔM51 or VSVM51R, respectively); (2) truncation of the C-terminus of the G protein (GΔ28). Despite these mutations, recombinant VSV with mutated M protein is only moderately attenuated in animals, whereas there are no detailed reports to determine the pathogenicity of recombinant VSV with truncated G protein at high dose. Thus, a novel recombinant VSV (VSVΔM51-GΔ28) as well as other attenuated VSVs (VSVΔM51, VSV-GΔ28) were produced to determine their efficacy as vaccine vectors with low pathogenicity. In vitro studies indicated that truncated G protein (GΔ28) could play a more important role than deletion of M51 (ΔM51) for attenuation of recombinant VSV. VSVΔM51-GΔ28 was determined to be the most attenuated virus with low pathogenicity in mice, with VSV-GΔ28 also showing relatively reduced pathogenicity. Further, neutralizing antibodies stimulated by VSV-GΔ28 proved to be significantly higher than in mice treated with VSVΔM51-GΔ28. In conclusion, among different attenuated VSVs with mutated M and/or G proteins, recombinant VSV with only truncated G protein (VSV-GΔ28) demonstrated ideal balance between pathogenesis and stimulating a protective immune response. These properties make VSV-GΔ28 a promising vaccine vector and vaccine candidate for preventing vesicular stomatitis disease.