E3 Ubiquitin Protein Ligase Research Articles

A circRNA therapy based on Rnf103 to inhibit Vibrio anguillarum infection

The losses caused by Vibrio infections in the aquaculture industry are challenging to quantify. In the face of antibiotic resistance, a natural and environmentally friendly alternative is urgently needed. In this study, we identify E3 ubiquitin-protein ligase RNF103 (rnf103) as a crucial target involved in immune evasion by Vibrio anguillarum. Our research demonstrates that Rnf103 promotes immune escape by inhibiting Traf6. Interestingly, we discover a circular RNA (circRNA), circRnf103, formed by reverse splicing of the Rnf103 gene. Predictive analysis and experimentation reveal that circRnf103 encodes Rnf103-177aa, a protein that competes with Rnf103 and binds to Traf6, preventing its degradation. Notably, circRnf103 therapy induces Rnf103-177aa protein production in zebrafish. In zebrafish models, circRnf103 exhibits significant effectiveness in treating V.anguillarum infections, reducing organ burden. These findings highlight the potential of circRNA therapy as a natural and innovative approach to combat infectious diseases sustainably, particularly in aquaculture and environmental management.

The HACE1 E3 ligase mediates RAC1-dependent control of mTOR signaling complexes.

HACE1 is a HECT family E3 ubiquitin-protein ligase with broad but incompletely understood tumor suppressor activity. Here, we report a previously unrecognized link between HACE1 and signaling complexes containing mammalian target of rapamycin (mTOR). HACE1 blocks mTORC1 and mTORC2 activities by reducing mTOR stability in an E3 ligase-dependent manner. Mechanistically, HACE1 binds to and ubiquitylates Ras-related C3 botulinum toxin substrate 1 (RAC1) when RAC1 is associated with mTOR complexes, including at focal adhesions, leading to proteasomal degradation of RAC1. This in turn decreases the stability of mTOR to reduce mTORC1 and mTORC2 activity. HACE1 deficient cells show enhanced mTORC1/2 activity, which is reversed by chemical or genetic RAC1 inactivation but not in cells expressing the HACE1-insensitive mutant, RAC1K147R . In vivo, Rac1 deletion reverses enhanced mTOR expression in KRasG12D -driven lung tumors of Hace1-/- mice. HACE1 co-localizes with mTOR and RAC1, resulting in RAC1-dependent loss of mTOR protein stability. Together, our data demonstrate that HACE1 destabilizes mTOR by targeting RAC1 within mTOR-associated complexes, revealing a unique ubiquitin-dependent process to control the activity of mTOR signaling complexes.

Generation of the iPSC line FINi002-A from a male Parkinson's disease patient carrying compound heterozygous mutations in the PRKN gene

The most common cause of autosomal recessive familial Parkinson’s disease (PD) are mutations in the PRKN/PARK2 gene encoding an E3 ubiquitin protein-ligase PARKIN. We report the generation of an iPSC cell line from the fibroblasts of a male PD patient carrying a common missense variant in exon 7 (p.Arg275Trp), and a 133 kb deletion encompassing exon 8, using transiently-present Sendai virus. The established line displays typical human primed iPSC morphology and expression of pluripotency-associated markers, normal karyotype without SNP array-detectable copy number variations and can give rise to derivatives of all three embryonic germ layers. We envisage the usefulness of this iPSC line, carrying a common and well-studied missense mutation in the RING1 domain of the PARKIN protein, for the elucidation of PARKIN-dependent mechanisms of PD using in vitro and in vivo models.

Cotadutide improves brown adipose tissue thermogenesis in obese mice

We studied the effect of cotadutide, a dual agonist glucagon-like peptide 1 (GLP1)/Glucagon, on interscapular brown adipose tissue (iBAT) remodeling and thermogenesis of obese mice. Twelve-week-old male C57BL/6 mice were fed a control diet (C group, n = 20) or a high-fat diet (HF group, n = 20) for ten weeks. Then, animals were redivided, adding cotadutide treatment: C, CC, HF, and HFC for four additional weeks. The multilocular brown adipocyte structure showed fat conversion (whitening), hypertrophy, and structural disarray in the HF group, which was reverted in cotadutide-treated animals. Cotadutide enhances the body temperature, thermogenesis, and sympathetic innervation (peroxisome proliferator-activated receptor-α, β3 adrenergic receptor, interleukin 6, and uncoupled protein 1), reduces pro-inflammatory markers (disintegrin and metallopeptidase domain, morphogenetic protein 8a, and neuregulin 4), and improves angiogenesis (vascular endothelial growth factor A, and perlecan). In addition, cotadutide enhances lipolysis (perilipin and cell death-inducing DNA fragmentation factor α), mitochondrial biogenesis (nuclear respiratory factor 1, transcription factor A mitochondrial, mitochondrial dynamin-like GTPase, and peroxisome proliferator-activated receptor gamma coactivator 1α), and mitochondrial fusion/fission (dynamin-related protein 1, mitochondrial fission protein 1, and parkin RBR E3 ubiquitin protein ligase). Cotadutide reduces endoplasmic reticulum stress (activating transcription factor 4, C/EBP homologous protein, and growth arrest and DNA-damage inducible), and extracellular matrix markers (lysyl oxidase, collagen type I α1, collagen type VI α3, matrix metallopeptidases 2 and 9, and hyaluronan synthases 1 and 2). In conclusion, the experimental evidence is compelling in demonstrating cotadutide's thermogenic effect on obese mice's iBAT, contributing to unraveling its action mechanisms and the possible translational benefits.

Genome-wide association study using a single-step approach for teat number in Duroc, Landrace and Yorkshire pigs in Korea.

We investigated the genetic basis of teat number in sows, which is an important factor in their reproductive performance. We collected genotyping data from 20 353 pigs of three breeds (Duroc, Landrace and Yorkshire) using the Porcine SNP60K Bead Chip, and analyzed phenotypic data from 240 603 pigs. The heritability values of total teat number were 0.33 ± 0.02, 0.51 ± 0.01 and 0.50 ± 0.01 in Duroc, Landrace and Yorkshire pigs, respectively. A genome-wide association study was used to identify significant chromosomal regions associated with teat number in SSC7 and SSC9 in Duroc pig, SSC3, SSC7 and SSC18 in Landrace pig, and SSC7, SSC8 and SSC10 in Yorkshire pig. Among the markers, MARC0038565, located between the vertnin (VRTN) and synapse differentiation-inducing 1-like (SYNDIG1L) genes, showed the strongest association in the Duroc pig and was significant in all breeds. In Landrace and Yorkshire pigs, the most significant markers were located within the apoptosis resistant E3 ubiquitin protein ligase 1 (AREL1) and latent transforming growth factor beta-binding protein 2 (LTBP2) genes in SSC7, respectively. VRTN is a candidate gene regulating the teat number. Most markers were located in SSC7, indicating their significance in determining teat number and their potential as valuable genomic selection targets for improving this trait. Extensive linkage disequilibrium blocks were identified in SSC7, supporting their use in genomic selection strategies. Our study provides valuable insights into the genetic architecture of teat numbers in pigs, and helps identify candidate genes and genomic regions that may contribute to this economically important trait.

Genome-wide association mapping for yield-related traits in soybean (Glycine max) under well-watered and drought-stressed conditions.

Soybean (Glycine max) productivity is significantly reduced by drought stress. Breeders are aiming to improve soybean grain yields both under well-watered (WW) and drought-stressed (DS) conditions, however, little is known about the genetic architecture of yield-related traits. Here, a panel of 188 soybean germplasm was used in a genome wide association study (GWAS) to identify single nucleotide polymorphism (SNP) markers linked to yield-related traits including pod number per plant (PN), biomass per plant (BM) and seed weight per plant (SW). The SLAF-seq genotyping was conducted on the population and three phenotype traits were examined in WW and DS conditions in four environments. Based on best linear unbiased prediction (BLUP) data and individual environmental analyses, 39 SNPs were significantly associated with three soybean traits under two conditions, which were tagged to 26 genomic regions by linkage disequilibrium (LD) analysis. Of these, six QTLs qPN-WW19.1, qPN-DS8.8, qBM-WW1, qBM-DS17.4, qSW-WW4 and qSW-DS8 were identified controlling PN, BM and SW of soybean. There were larger proportions of favorable haplotypes for locus qPN-WW19.1 and qSW-WW4 rather than qBM-WW1, qBM-DS17.4, qPN-DS8.8 and qSW-DS8 in both landraces and improved cultivars. In addition, several putative candidate genes such as Glyma.19G211300, Glyma.17G057100 and Glyma.04G124800, encoding E3 ubiquitin-protein ligase BAH1, WRKY transcription factor 11 and protein zinc induced facilitator-like 1, respectively, were predicted. We propose that the further exploration of these locus will facilitate accelerating breeding for high-yield soybean cultivars.

OR07-01 Aberrant Expression Of Agouti Signaling Protein (ASIP) As A Cause of Monogenic Severe Childhood Obesity

Abstract Disclosure: K. Landgraf: None. E.T. Kempf: None. R. Stein: None. M. Hanschkow: None. Y. Tseng: None. T. Schöneberg: None. E. Zeggini: None. M. Blüher: None. A. Körner: None. Here we identified and characterized a heterozygous tandem duplication at the ASIP (agouti-signaling protein) gene locus causing ubiquitous, ectopic ASIP expression in a female index patient with extreme childhood obesity.From an adipose tissue sample of the patient, we observed pronounced and sustained differentiation potential of stroma-vascular fraction (SVF) cells into adipocytes in the patient compared to normal control cells. We further found reduced mitochondrial maximum respiration, spare capacity, and proton leak in the patient’s SVF cells as proxies for reduced energy expenditure. By transcriptome screen, we identified overexpression of one single gene, ASIP. We found and functionally confirmed a chromosomal rearrangement at the ASIP locus, that places ASIP under control of the ubiquitously active itchy E3 ubiquitin protein ligase (ITCH) promoter. The patient's phenotype of early-onset obesity, overgrowth, red hair, and hyperinsulinemia is concordant with that of mutant, so called agouti mice ubiquitously expressing the homolog nonagouti. Physiologically, ASIP antagonises the melanocortin 1 receptor (MC1R) in the skin. Due to its homology to the hypothalamic regulator of eating behaviour agouti-related protein (AGRP), we hypothesize that ectopic ASIP similarly influences eating behaviour via repressing melanocyte-stimulating hormone-mediated activation of MC4R and that the mutation might therefore represent a novel monogenic cause for severe childhood obesity. We show that the tandem duplication results in the ectopic generation of ASIP in patient-derived native stroma-vascular fraction (SVF) cells, adipocytes and blood cells as well as in induced pluripotent stem cells (iPSCs) generated from SVF cells of the patient, thus indicating ubiquitous ectopic ASIP expression. This overexpression persisted after differentiation of iPSCs into the three germ layers, mesoderm, ectoderm and endoderm, and after differentiation into hypothalamic-like neurons. Furthermore, we observed that ASIP causes a reduction of MC4R activity in vitro supporting the hypothesis that the obesity phenotype of our patient is caused by ectopic ASIP acting at the MC4R in the hypothalamus. Since the type of mutation escapes standard genetic screening algorithms, we rescreened the Leipzig childhood obesity cohort of 1745 patients and identified four additional patients with the identical mutation, ectopic ASIP expression and a similar phenotype. Taken together, our data indicate that ubiquitous ectopic ASIP expression is likely a monogenic cause of human obesity and might be potentially treatable with melanocortin 4 receptor agonists. Presentation: Thursday, June 15, 2023

CircRNA itchy E3 ubiquitin protein ligase improves mitochondrial dysfunction in sepsis-induced acute kidney injury by targeting microRNA-214-3p/ATP-binding cassette A1 axis

Background Circular RNAs (circRNAs) are promising biomarkers and therapeutic targets for acute kidney injury (AKI). In this study, we investigated the mechanism by which circRNA itchy E3 ubiquitin protein ligase (circ-ITCH) regulates sepsis-induced AKI. Methods A sepsis-induced AKI mouse model was created using LPS induction and circ-ITCH overexpression. Circ-ITCH levels were confirmed via RT-qPCR. Kidney tissue changes were examined through various stains and TUNEL. Enzyme-linked immunosorbent assay (ELISA) gauged oxidative stress and inflammation. Mitochondrial features were studied with electron microscopy. RT-qPCR and western blotting assessed mitochondrial function parameters. Using starBase, binding sites between circ-ITCH and miR-214-3p, as well as miR-214-3p and ABCA1, were predicted. Regulatory connections were proven by dual-luciferase assay, RT-qPCR, and western blotting. Results Circ-ITCH expression was downregulated in LPS-induced sepsis mice. Overexpression of circ-ITCH ameliorates indicators of renal function (serum creatinine [SCr], blood urea nitrogen [BUN], neutrophil gelatinase-associated lipocalin [NGAL], and kidney injury molecule-1 [Kim-1]), reduces renal cell apoptosis, mitigates oxidative stress markers (reactive oxygen species [ROS] and malondialdehyde [MDA]), and diminishes inflammatory markers (interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF-α]). Moreover, circ-ITCH overexpression alleviated mitochondrial damage and dysfunction. Furthermore, circ-ITCH acts as a sponge for miR-214-3p, thereby upregulating ABCA1 expression. In addition, the miR-214-3p inhibitor repressed oxidative stress, inflammation, and mitochondrial dysfunction, which was reversed by circ-ITCH knockdown. Further cellular analysis in HK-2 cells supported these findings, highlighting the protective role of circ-ITCH against sepsis-induced AKI, particularly through the miR-214-3p/ABCA1 axis. Conclusion The novel circ-ITCH/miR-214-3p/ABCA1 pathway plays an essential role in the regulation of oxidative stress and mitochondrial dysfunction in sepsis-induced AKI.

NEDD4 like E3 ubiquitin protein ligase represses astrocyte activation and aggravates neuroinflammation in mice with depression via paired box 6 ubiquitination.

Astrocytes are implicated in stress-induced neuroinflammatory responses in depression. This paper was to explore the molecular mechanism of the E3 ubiquitin ligase NEDD4L (NEDD4 like E3 ubiquitin protein ligase) in depressed mice by regulating astrocyte activation, and to find a new target for depression. A mouse model of depression was established by CUMS (chronic mild unpredictable stress) in 48 6-week male C57BL/6 mice and injected with sh-NEDD4L vector for testing behavioral and cognitive abilities, histopathological changes, and the number of GFAP-positive cells. The mRNA and protein levels of NEDD4L, PAX6 (paired box 6) and P2X7R (purinergic ligand-gated ion channel 7 receptor) were measured. Inflammation model was established by lipopolysaccharide treatment of mouse astrocyte line C8-D1A and infected with sh-NEDD4L. After CUMS induction, mice showed depression-like symptoms, increased inflammatory infiltration, decreased glial fibrillary acidic protein (GFAP)-positive cells in brain tissue, and increased NEDD4L protein levels. NEDD4L inhibition increased GFAP-positive cells, increased PAX6 protein levels and decreased P2X7R mRNA and protein levels, and decreased inflammatory factor secretion in brain tissue and in vitro cells. PAX6 knockdown or P2X7R overexpression partially reversed the effects of NEDD4L inhibition on astrocyte activation and neuroinflammation. To conclude, highly-expressed NEDD4L in depression-like mouse brain inhibits astrocyte activation and exacerbates neuroinflammation by ubiquitinating PAX6 and promoting P2X7R level.

SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

Transforming growth factor-β (TGF-β) is the primary factor that drives fibrosis in most, if not all, forms of chronic kidney disease. In kidneys that are obstructed, specific deletion of Sirt2 in renal tubule epithelial cells (TEC) has been shown to aggravate renal fibrosis, while renal tubule specific overexpression of Sirt2 has been shown to ameliorate renal fibrosis. Similarly, specific deletion of Sirt2 in hepatocyte aggravated CCl4-induced hepatic fibrosis. In addition, we have demonstrated that SIRT2 overexpression and knockdown restrain and enhance TGF-β-induced fibrotic gene expression, respectively, in TEC. Mechanistically, SIRT2 reduced the phosphorylation, acetylation, and nuclear localization levels of SMAD2 and SMAD3, leading to inhibition of the TGF-β signaling pathway. Further studies have revealed that that SIRT2 was able to directly interact with and deacetylate SMAD2 at lysine 451, promoting its ubiquitination and degradation. Notably, loss of SMAD specific E3 ubiquitin protein ligase 2 abolishes the ubiquitination and degradation of SMAD2 induced by SIRT2 in SMAD2. Regarding SMAD3, we have found that SIRT2 interact with and deacetylates SMAD3 at lysine 341 and 378 only in the presence of TGF-β, thereby reducing its activation. This study provides initial indication of the anti-fibrotic role of SIRT2 in renal tubules and hepatocytes, suggesting its therapeutic potential for fibrosis.

Intercellular adhesion molecule 2 as a novel prospective tumor suppressor induced by ERG promotes ubiquitination-mediated radixin degradation to inhibit gastric cancer tumorigenicity and metastasis

BackgroundGastric cancer (GC) is a fatal cancer with unclear pathogenesis. In this study, we explored the function and potential mechanisms of intercellular adhesion molecule 2 (ICAM2) in the development and advancement of GC.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to quantify ICAM2 expression in harvested GC tissues and cultured cell lines. Immunohistochemical analyses were conducted on a GC tissue microarray to quantify ICAM2 expression and explore its implication on the prognosis of GC patients. In vitro experiments were carried out to reveal the biological functions of ICAM2 in GC cell lines. Further, in vivo experiments were conducted using xenograft models to assess the impact of ICAM2 on GC development and metastasis. Western blot, immunofluorescence, immunoprecipitation, luciferase assay, chromatin immunoprecipitation, and ubiquitination analysis were employed to investigate the underlying mechanisms.ResultsICAM2 expression was downregulated in GC, positively correlating with advanced T stage, distant metastasis, advanced clinical stage, vessel invasion, and shorter patient survival time. ICAM2 overexpression suppressed the proliferation, migration, invasion, metastasis of GC cells as well as their ability to form tumors, whereas ICAM2 knockdown yielded opposite results. Erythroblast transformation-specific-related gene (ERG) as a transcription factor promoted the transcription of ICAM2 by binding to the crucial response element localized within its promoter region. Further analysis revealed that ICAM2 reduced radixin (RDX) protein stability and expression. In these cells, ICAM2 bound to the RDX protein to promote the ubiquitination and degradation of RDX via NEDD4 Like E3 Ubiquitin Protein Ligase (NEDD4L), and this post-translational modification resulted in the inhibition of GC.ConclusionsIn summary, this study demonstrates that ICAM2, which is induced by ERG, suppresses GC progression by enhancing the ubiquitination and degradation of RDX in a NEDD4L-dependent manner. Therefore, these results suggest that ICAM2 is a potential prognostic marker and a therapeutic target for GC.

FBXL5 promotes lipid accumulation in alcoholic fatty liver disease by promoting the ubiquitination and degradation of TFEB

BackgroundAlcoholic fatty liver disease (AFLD) is characterized by abnormal lipid droplet accumulation in liver. Epigenetic regulation plays an important role in the pathogenesis of AFLD. Comprehensive bioinformatics analysis revealed that an E3 ubiquitin ligase, F-box and leucine-rich repeats protein 5 (FBXL5), was significantly upregulated in AFLD mice. MethodsThe mouse model of AFLD was established by feeding Lieber-DeCarli liquid diet containing ethanol. An in vitro model of AFLD was established by treating HepG2 cells with ethanol (EtOH). The FBXL5 expression was assessed by quantitative real-time PCR (qRT-PCR) and western blotting assays. The levels of triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lipid accumulation were analyzed by enzyme-linked immunosorbent assay (ELISA) and Nile red staining. ResultsThe FBXL5 expression was markedly up-regulated in in vivo and in vitro models of AFLD compared with controls. Functionally, FBXL5 knockdown alleviated lipid accumulation in EtOH-treated HepG2 cells. Mechanistically, FBXL5 directly interacted with transcription factor EB (TFEB) and accelerated its ubiquitination-mediated degradation. TFEB knockdown reversed the effect of FBXL5 inhibition on decreasing EtOH-induced lipid accumulation. ConclusionOur data suggest that FBXL5 promotes lipid accumulation in AFLD by promoting the ubiquitination and degradation of TFEB.

Osteopontin regulates the growth and invasion of liver cancer cells via DTL.

Osteopontin (OPN), a secreted phosphoglycoprotein, has important roles in tumor growth, invasion and metastasis in numerous types of cancers. Denticleless E3 ubiquitin protein ligase homolog (DTL), one of the CUL4-DDB1-associated factors (DCAFs), has also been associated with the invasion and metastasis of cancer cells. In the present study, OPN was found to induce DTL expression in liver cancer cells, and the results obtained using luciferase activity assays demonstrated that OPN could transcriptionally activate DTL expression in liver cancer cells. Furthermore, the results of the present study demonstrated that OPN could increase the expression of DTL via PI3K/AKT signaling. In conclusion, the present study demonstrated that OPN, as an extracellular matrix protein, is able to promote the growth and invasion of liver cancer cells through stimulation of the expression of DTL via the PI3K/AKT signaling pathway.

Abstract P218: Ubr1 And Nedd4-2 Synergistically Modulate Ace2 Ubiquitination In Hypertensive Male Mice

Angiotensin-converting enzyme 2 (ACE2) is a counter-regulatory player in the renin-angiotensin system (RAS). Downregulation of ACE2 in hypertension results in vasoconstriction, sympathetic over-activation, and hypertension. Recently, we identified UBR1, an E3 ubiquitin-protein ligase, as a potential ubiquitinase modulating ACE2 expression, notably in hypertensive male mice where UBR1 is upregulated in the hypothalamus and peripheral organs. Here, we aim to understand the role of UBR1 in hypertension and its crosstalk with Nedd4-2, another E3 ligase regulating ACE2. C57BI/6J male mice (3 month-old, n=18) were implanted with telemetry probes for weekly 24 h BP recording. One week following baseline BP recording, mice were implanted with subcutaneous osmotic pumps containing Ang-II (600 ng/kg/min). After confirming hypertension, mice were divided into 4 treatment groups (n=6/group); 1) peripheral UBR1 siRNA via osmotic pump (50 μg/day/2 weeks), 2) central UBR1 siRNA via intracerebroventricular infusion (ICV) (50 μg/day/2 weeks) 3) scrambled shRNA + Ang-II, and 4) no treatment. After 6 weeks of BP recording, mice were euthanized, and hypothalami were harvested for protein analysis (Capillary Western). BP was transiently reduced during the resting phase after 3 weeks of ICV UBR1 siRNA infusion compared to the Ang-II group (MAP: 114±7 vs . 135±3 mmHg). Ang-II infusion enhanced UBR1 protein levels versus no treatment (Fold Change:1.6±0.1 vs . 1.0±0.04; p<0.05) which was prevented by peripheral and central UBR1 siRNA infusion, respectively (0.7±0.08, 1.3±0.08 vs. 1.6±0.1, p<0.05). Interestingly, while UBR1 siRNA infusion increased ACE2 expression (1.7±0.1, 2±0.2, p<0.01, respectively), it was also associated with increased Nedd4-2 levels ( 2.9±0.1, 3.5±0.2, p<0.0001, respectively), and pNedd4-2 (4.1±0.4, 3.8±0.5, p<0.001, respectively). Together, these data suggest that while prevention of UBR1 upregulation in hypertension is associated with an increase in ACE2 expression, it is not sufficient to permanently reduce BP as other E3 ligases, like Nedd4-2, appear to compensate for the knockdown.

CircMIB2 therapy can effectively treat pathogenic infection by encoding a novel protein

The mRNA therapy is widely used in the treatment of diseases due to its efficient characteristics, and the COVID-19 vaccine is the application of mRNA therapy. However, due to the instability of mRNA, mRNA vaccines often need lots of modifications to ensure its stability. Recent research shows that circRNA with stable RNA structure can encode protein, which provides a new direction for mRNA therapy. Here, we discovered a novel circRNA (circMIB2) derived from E3 ubiquitin-protein ligase MIB2 (MIB2) gene in lower vertebrate fish, which can translate into a 134 amino acid protein (MIB2-134aa) through m6A modification, and is involved in innate immunity. MIB2-134aa is completely consistent with the amino acid sequence of the two domains of host gene MIB2 protein; host gene MIB2 can target TRAF6 through the two domains and inhibit the innate immune response by promoting the ubiquitination degradation of the K11-link of TRAF6, MIB2-134aa also targets TRAF6 through these same domains. Interestingly, MIB2-134aa greatly reduced the degradation of TRAF6 by its host gene MIB2. More importantly, we found that circRNA therapy of circMIB2 can significantly inhibit the colonization of Vibrio anguillarum in zebrafish, and it provides a new direction for the treatment of pathogenic diseases of fish.

Circ-UBR4 regulates the proliferation, migration, inflammation, and apoptosis in ox-LDL-induced vascular smooth muscle cells via miR-515-5p/IGF2 axis.

The aim of our study is to disclose the role and underlying molecular mechanisms of circular RNA ubiquitin protein ligase E3 component n-recognin 4 (circ-UBR4) in atherosclerosis (AS). Our data showed that circ-UBR4 expression was upregulated in AS patients and oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cells (VSMCs) compared with healthy volunteer and untreated VSMCs. In addition, ox-LDL stimulated proliferation, migration, and inflammation but decreased apoptosis in VSMCs, which were overturned by the inhibition of circ-UBR4. miR-515-5p was sponged by circ-UBR4, and its inhibitor reversed the inhibitory effect of circ-UBR4 knockdown on proliferation, migration, and inflammation in ox-LDL-induced VSMCs. Insulin-like growth factor2 (IGF2) was a functional target of miR-515-5p, and overexpression of IGF2 reversed the suppressive effect of miR-515-5p on ox-LDL-stimulated VSMCs proliferation, migration, and inflammation. Collectively, circ-UBR4 knockdown decreased proliferation, migration, and inflammation but stimulated apoptosis in ox-LDL-induced VSMCs by targeting the miR-515-5p/IGF2 axis.

SP1-Induced Upregulation of LncRNA AFAP1-AS1 Promotes Tumor Progression in Triple-Negative Breast Cancer by Regulating mTOR Pathway.

The long non-coding RNA (lncRNA) actin fiber-associated protein-1 antisense RNA 1 (AFAP1-AS1) exerted oncogenic activity in triple-negative breast cancer (TNBC). We designed this study and conducted it to investigate the upstream regulation mechanism of AFAP1-AS1 in TNBC tumorigenesis. In this work, we proved the localization of AFAP1-AS1 in the cytoplasm. We elucidated the mechanism by which the transcription factor specificity protein 1 (SP1) modulated AFAP1-AS1 in TNBC progression, which has yet to be thoroughly studied. Dual luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay revealed a strong affinity of SP1 toward the promoter regions P3 of AFAP1-AS1, proving the gene expression regulation of AFAP1-AS1 via SP1 in TNBC. Additionally, SP1 could facilitate the tumorigenesis of TNBC cells in vitro and in vivo by regulating the AFAP1-AS1 expression. Furthermore, silenced AFAP1-AS1 suppressed the expression of genes in the mTOR pathway, such as eukaryotic translation initiation factor 4B (EIF4B), mitogen-activated protein kinase-associated protein 1 (MAPKAP1), SEH1-like nucleoporin (SEH1L), serum/glucocorticoid regulated kinase 1 (SGK1), and its target NEDD4-like E3 ubiquitin protein ligase (NEDD4L), and promoted the gene expression of s-phase kinase-associated protein 2 (SKP2). Overall, this study emphasized the oncogenic role of SP1 and AFAP1-AS1 in TNBC and illustrated the AFAP1-AS1 upstream interaction with SP1 and the downstream modulatory of mTOR signaling, thus offering insights into the tumorigenesis mechanism in TNBC.

Genome-Wide Survey of the RWP-RK Gene Family in Cassava (Manihot esculenta Crantz) and Functional Analysis.

The plant-specific RWP-RK transcription factor family plays a central role in the regulation of nitrogen response and gametophyte development. However, little information is available regarding the evolutionary relationships and characteristics of the RWP-RK family genes in cassava, an important tropical crop. Herein, 13 RWP-RK proteins identified in cassava were unevenly distributed across 9 of the 18 chromosomes (Chr), and these proteins were divided into two clusters based on their phylogenetic distance. The NLP subfamily contained seven cassava proteins including GAF, RWP-RK, and PB1 domains; the RKD subfamily contained six cassava proteins including the RWP-RK domain. Genes of the NLP subfamily had a longer sequence and more introns than the RKD subfamily. A large number of hormone- and stress-related cis-acting elements were found in the analysis of RWP-RK promoters. Real-time quantitative PCR revealed that all MeNLP1-7 and MeRKD1/3/5 genes responded to different abiotic stressors (water deficit, cold temperature, mannitol, polyethylene glycol, NaCl, and H2O2), hormonal treatments (abscisic acid and methyl jasmonate), and nitrogen starvation. MeNLP3/4/5/6/7 and MeRKD3/5, which can quickly and efficiently respond to different stresses, were found to be important candidate genes for further functional assays in cassava. The MeRKD5 and MeNLP6 proteins were localized to the cell nucleus in tobacco leaf. Five and one candidate proteins interacting with MeRKD5 and MeNLP6, respectively, were screened from the cassava nitrogen starvation library, including agamous-like mads-box protein AGL14, metallothionein 2, Zine finger FYVE domain containing protein, glyceraldehyde-3-phosphate dehydrogenase, E3 Ubiquitin-protein ligase HUWE1, and PPR repeat family protein. These results provided a solid basis to understand abiotic stress responses and signal transduction mediated by RWP-RK genes in cassava.

High-density lipoprotein regulates angiogenesis by long non-coding RNA HDRACA

Normal high-density lipoprotein (nHDL) can induce angiogenesis in healthy individuals. However, HDL from patients with coronary artery disease undergoes various modifications, becomes dysfunctional (dHDL), and loses its ability to promote angiogenesis. Here, we identified a long non-coding RNA, HDRACA, that is involved in the regulation of angiogenesis by HDL. In this study, we showed that nHDL downregulates the expression of HDRACA in endothelial cells by activating WW domain-containing E3 ubiquitin protein ligase 2, which catalyzes the ubiquitination and subsequent degradation of its transcription factor, Kruppel-like factor 5, via sphingosine 1-phosphate (S1P) receptor 1. In contrast, dHDL with lower levels of S1P than nHDL were much less effective in decreasing the expression of HDRACA. HDRACA was able to bind to Ras-interacting protein 1 (RAIN) to hinder the interaction between RAIN and vigilin, which led to an increase in the binding between the vigilin protein and proliferating cell nuclear antigen (PCNA) mRNA, resulting in a decrease in the expression of PCNA and inhibition of angiogenesis. The expression of human HDRACA in a hindlimb ischemia mouse model inhibited the recovery of angiogenesis. Taken together, these findings suggest that HDRACA is involved in the HDL regulation of angiogenesis, which nHDL inhibits the expression of HDRACA to induce angiogenesis, and that dHDL is much less effective in inhibiting HDRACA expression, which provides an explanation for the decreased ability of dHDL to stimulate angiogenesis.

Differentially Expressed Genes in Response to a Squalene-Supplemented Diet Are Accurate Discriminants of Porcine Non-Alcoholic Steatohepatitis.

Squalene is the major unsaponifiable component of virgin olive oil, the fat source of the Mediterranean diet. To evaluate its effect on the hepatic transcriptome, RNA sequencing was carried out in two groups of male Large White x Landrace pigs developing nonalcoholic steatohepatitis by feeding them a high fat/cholesterol/fructose and methionine and choline-deficient steatotic diet or the same diet with 0.5% squalene. Hepatic lipids, squalene content, steatosis, activity (ballooning + inflammation), and SAF (steatosis + activity + fibrosis) scores were analyzed. Pigs receiving the latter diet showed hepatic squalene accumulation and twelve significantly differentially expressed hepatic genes (log2 fold change < 1.5 or <1.5) correlating in a gene network. These pigs also had lower hepatic triglycerides and lipid droplet areas and higher cellular ballooning. Glutamyl aminopeptidase (ENPEP) was correlated with triglyceride content, while alpha-fetoprotein (AFP), neutralized E3 ubiquitin protein ligase 3 (NEURL3), 2'-5'-oligoadenylate synthase-like protein (OASL), and protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B) were correlated with activity reflecting inflammation and ballooning, and NEURL3 with the SAF score. AFP, ENPEP, and PPP1R1B exhibited a remarkably strong discriminant power compared to those pathological parameters in both experimental groups. Moreover, the expression of PPP1R1B, TMEM45B, AFP, and ENPEP followed the same pattern in vitro using human hepatoma (HEPG2) and mouse liver 12 (AML12) cell lines incubated with squalene, indicating a direct effect of squalene on these expressions. These findings suggest that squalene accumulated in the liver is able to modulate gene expression changes that may influence the progression of non-alcoholic steatohepatitis.