Ubiquitin-mediated Proteolysis Research Articles

Transcriptome analysis and identification of genes associated with leaf crude protein content in foxtail millet [Setaria italica (L.) P. Beauv.

Introduction: Spruce spider mite is a primary insect pest of Chinese chestnut in China and seriously influences its yield and quality. However, the current management against this mite is costly and poorly effective. In previous research, we bred several foxtail millet materials for interplanting with chestnut tree, and found that they had high levels of crude protein (CP) in leaves and attracted spruce spider mite to feed on the leaves, thereby reducing chestnut damage. Methods: In this study, four foxtail millet varieties with significant differences in leaf crude protein content were used for high-throughput sequencing and identification of genes associated with leaf crude protein content. Gene enrichment analyses were carried out to comprehend the functions of these genes and the biological processes in which they are involved. In addition, transcription factors (TFs) were evaluated. Results: 435 differentially expressed genes (DEGs) were identified, suggesting their potential role in crude protein accumulation. Some differentially expressed genes were found to be associated with nitrogen metabolism and ubiquitin-mediated proteolysis pathways. Moreover, we identified 40 TF genes categorized into 11 transcription factor families. Discussion: Our findings represent an important resource that clarifies the mechanisms of accumulation and control of leaf crude protein in foxtail millet, and provide an opportunity for suppression of spruce spider mite attack on Chinese chestnut by interplanting with foxtail millet varieties with high concentrations of leaf crude protein.

RNA sequencing and bioinformatics analysis of circular RNAs in asphyxial newborns with acute kidney injury.

As one kind of novel noncoding RNA, circular RNAs (circRNAs) are involved in different biological processes. Although growing evidences have supported the important role of circRNAs in renal diseases, the mechanism remains unclear in neonatal acute kidney injury (AKI). High-throughput sequencing analysis was used to investigate the expression of circRNAs between hypoxia-induced AKI neonates and controls. Bioinformatics analysis was conducted to predict the function of differentially expressed circRNAs. Finally, the differentially expressed circRNAs were screened and determined by quantitative real-time PCR (qPCR). (1) A total of 296 differentially expressed circRNAs were identified (Fold change >2 and p < 0.05). Of them, 184 circRNAs were markedly upregulated, and 112 were significantly downregulated in the AKI group. (2) The pathway analysis showed that ubiquitin-mediated proteolysis, renal cell carcinoma, Jak-STAT, and HIF-1 signaling pathways participated in AKI. (3) Top five upregulated and five downregulated circRNAs with higher fold changes were selected for qPCR validation. Hsa_circ_0008898 (Fold Change=5.48, p=0.0376) and hsa_circ_0005519 (Fold Change=4.65, p=0.0071) were significantly upregulated, while hsa_circ_0132279 (Fold Change=-4.47, p=0.0008), hsa_circ_0112327 (Fold Change=-4.26, p=0.0048), and hsa_circ_0017647 (Fold Change=-4.15, p=0.0313) were significantly downregulated in asphyxia-induced AKI group compared with the control group. This study could contribute to future research on neonatal AKI and facilitate the identification of novel therapeutic targets.

Toxicologic effect of short-term enrofloxacin exposure on brain of Carassius auratus var. Pengze

To further explore short-term exposure of enrofloxacin (ENR) induced toxicity in crucian carp brain that has been reported by our previous work, as well as the possible toxicological mechanisms, this study investigated the blood-brain barrier (BBB) permeability to low dosage of ENR through comprehensively assessing expression of BBB constitutive molecules zonula occludens-1 (ZO-1) and permeability glycoprotein (P-gp), as well as ENR residue in brain of crucian carp. Toxicologic effect of ENR on brain tissue was determined through evaluating expression of brain-derived proteins S100B, neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) in crucian carp brain tissue, as well as contents of the proteins in serum. The toxicological mechanisms were explored through analyzing transcriptome analysis data. Results showed that ENR possessed excellent permeability to crucian carp BBB, which was closely related to deranged BBB structure and declined ENR efflux that were attributed to downregulated expression of ZO-1 and P-gp by ENR exposure. Meanwhile, S100B, NSE and GFAP were upregulated in brain by ENR, and came out into blood across the damaged BBB. These data revealed that ENR induced disruption of BBB and damage of brain tissue in crucian carp. Transcriptome analysis data indicated that ENR induced toxicologic effect might be related to modification of metabolism, organismal systems, and genetic information processing, etc., and that PI3K/Akt, MAPK, HIF-1, and ubiquitin mediated proteolysis involved the mechanisms, most of the mechanisms were attributed to ENR induced oxidative stress in crucian carp brain.

CircRNA-miRNA-mRNA network targets ubiquitin-mediated proteolysis pathway in coronary artery disease

The convoluted crosstalk of non-coding RNA (ncRNA) and mRNA results in the regulation of pathogenic processes in coronary artery disease (CAD). We utilized an in silico approach to assess circular RNA (circRNA), microRNA (miRNA), and mRNA, as potential biomarkers for CAD. CircRNA pertaining to CAD, atherosclerosis, and myocardial infarction were extracted from two databases namely Circad and circR2Disease. miRNA target sites & RNA-binding protein interactions were predicted using the circRNAinteractome database. miRNA gene targets were predicted using TargetScan, DIANA, and miRDIP. Gene enrichment analysis revealed that circRNA-miRNA distinctively targets ubiquitin-mediated proteolysis pathways (p = 0.02). Dysregulated ubiquitin pathway is known to play a role in atherosclerotic plaque progression and instability.

Genetic Predisposition to Hepatocellular Carcinoma.

Liver preneoplastic and neoplastic lesions of the genetically susceptible F344 and resistant BN rats cluster, respectively, with human HCC with better (HCCB) and poorer prognosis (HCCP); therefore, they represent a valid model to study the molecular alterations determining the genetic predisposition to HCC and the response to therapy. The ubiquitin-mediated proteolysis of ERK-inhibitor DUSP1, which characterizes HCC progression, favors the unrestrained ERK activity. DUSP1 represents a valuable prognostic marker, and ERK, CKS1, or SKP2 are potential therapeutic targets for human HCC. In DN (dysplastic nodule) and HCC of F344 rats and human HCCP, DUSP1 downregulation and ERK1/2 overexpression sustain SKP2-CKS1 activity through FOXM1, the expression of which is associated with a susceptible phenotype. SAM-methyl-transferase reactions and SAM/SAH ratio are regulated by GNMT. In addition, GNMT binds to CYP1A, PARP1, and NFKB and PREX2 gene promoters. MYBL2 upregulation deregulates cell cycle and induces the progression of premalignant and malignant liver. During HCC progression, the MYBL2 transcription factor positively correlates with cells proliferation and microvessel density, while it is negatively correlated to apoptosis. Hierarchical supervised analysis, regarding 6132 genes common to human and rat liver, showed a gene expression pattern common to normal liver of both strains and BN nodules, and a second pattern is observed in F344 nodules and HCC of both strains. Comparative genetics studies showed that DNs of BN rats cluster with human HCCB, while F344 DNs and HCCs cluster with HCCP.

Identification of key programmed cell death-related genes and immune infiltration in extracorporeal membrane oxygenation treatment for acute myocardial infarction based on bioinformatics analysis.

Extracorporeal membrane oxygenation (ECMO) is an important clinical treatment for acute myocardial infarction (AMI) combined with cardiogenic shock, but the role of programmed cell death (PCD)-related genes in prognostication has not yet been investigated. Therefore, we explored the key prognostic biomarkers and immune infiltration in ECMO treatment in AMI combined with cardiogenic shock. The GSE93101 dataset was analyzed from the Gene Expression Omnibus (GEO) database, and the expression levels of PCD-related genes in AMI under ECMO were identified. Differentially expressed PCD-related genes between successful and failed treatment samples were analyzed, and Least absolute shrinkage and selection operator (LASSO) logistic regression and random forest were used to screen PCD-related molecular markers for ECMO treatment in AMI combined with cardiogenic shock. Co-expressed regulatory network and enrichment functions of the hub PCD-related genes were performed. In addition, the single-sample gene set enrichment analysis (ssGSEA) algorithm was used to calculate the immune cell infiltration of the ECMO treatment samples. A total of 115 differentially expressed genes were identified from the GSE93101 dataset, and 76 genes were associated with PCD. Then, two hub PCD-related genes, Cell division cycle associated 7 (CDCA7), ankyrin repeat and SOCS box containing 13 (ASB13) were identified as prognostic markers of ECMO treatment in AMI combined with cardiogenic shock. The most significant Gene Ontology (GO) enriched terms of the co-expressed protein of ASB13 are related to post-translational protein modification, cullin-RING ubiquitin ligase complex, and cullin family protein binding, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that ubiquitin mediated proteolysis is the most enriched pathway. The results of GO and KEGG analysis in CDCA7 were mainly involved in DNA and cell cycle related activities and pathways. Moreover, we found that the successful treatment samples contained a lower proportion of nature killer T cells using immune infiltration analysis. Immune cell infiltration analysis revealed that ASB13 was positively correlated with natural killer cell (r = 0.591, p = 0.026), monocyte (r = 0.586, p = 0.028), and gamma delta T cell (r = 0.562, p = 0.036). The results of this study showed that ASB13 and CDCA7 may contribute to the occurrence and progression of AMI with cardiogenic shock under ECMO.

The regulation role and diagnostic value of fibrinogen-like protein 1 revealed by pan-cancer analysis.

Although the role of fibrinogen-like protein 1 (FGL1) in tumorigenesis is well known, a pan-cancer analysis of FGL1 lacks. We used bioinformatics techniques to analyze cancer data from publicly available datasets from The Cancer Genome Atlas, UALCAN, TIMER, Gene Expression Profiling Interactive Analysis, cBioPortal, Search Tool for the Retrieval of Interacting Genes, and DAVID. FGL1 expression was significantly regulated in various common tumors than in normal tissues; it was increased in lung adenocarcinoma and decreased in colon adenocarcinoma. Cox regression analysis demonstrated that the upregulation of FGL1 expression was correlated with poor overall survival (OS) and disease-free survival (DFS) in stomach adenocarcinoma, brain low-grade glioma, cervical squamous cell carcinoma, and endocervical adenocarcinoma. Decreased FGL1 methylation levels were observed in majority of tumor types. FGL1 expression was significantly associated with the levels of immune cell subtypes and immune checkpoint genes. Deep deletion was the most common genetic mutation in FGL1 that led to frame-shift mutations, which was closely associated with poor progression-free interval, disease-specific survival, and OS in patients with FGL1 mutations. Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that FGL1-related genes participate in diverse pathways. Ubiquitin-mediated proteolysis is significantly correlated to the function of FGL1, which was identified for the first time in the present study. This pan-cancer study provides a deep understanding of the functions of FGL1 in progression of many tumors and demonstrates that FGL1 may be a potential biomarker for the diagnosis, prognosis, and immune infiltration in cancer.

Rice OsPUB16 modulates the 'SAPK9-OsMADS23-OsAOC' pathway to reduce plant water-deficit tolerance by repressing ABA and JA biosynthesis.

Ubiquitin-mediated proteolysis plays crucial roles in plant responses to environmental stress. However, the mechanism by which E3 ubiquitin ligases modulate plant stress response still needs to be elucidated. In this study, we found that rice PLANT U-BOX PROTEIN 16 (OsPUB16), a U-box E3 ubiquitin ligase, negatively regulates rice drought response. Loss-of-function mutants of OsPUB16 generated through CRISPR/Cas9 system exhibited the markedly enhanced water-deficit tolerance, while OsPUB16 overexpression lines were hypersensitive to water deficit stress. Moreover, OsPUB16 negatively regulated ABA and JA response, and ospub16 mutants produced more endogenous ABA and JA than wild type when exposed to water deficit. Mechanistic investigations revealed that OsPUB16 mediated the ubiquitination and degradation of OsMADS23, which is the substrate of OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 9 (SAPK9) and increases rice drought tolerance by promoting ABA biosynthesis. Further, the ChIP-qPCR analysis and transient transactivation activity assays demonstrated that OsMADS23 activated the expression of JA-biosynthetic gene OsAOC by binding to its promoter. Interestingly, SAPK9-mediated phosphorylation on OsMADS23 reduced its ubiquitination level by interfering with the OsPUB16-OsMADS23 interaction, which thus enhanced OsMADS23 stability and promoted OsAOC expression. Collectively, our findings establish that OsPUB16 reduces plant water-deficit tolerance by modulating the 'SAPK9-OsMADS23-OsAOC' pathway to repress ABA and JA biosynthesis.

Involvement of Small Non-Coding RNA and Cell Antigens in Pathogenesis of Extramedullary Multiple Myeloma

Extramedullary multiple myeloma (EMD) is an aggressive disease; malignant plasma cells lose their dependence in the bone marrow microenvironment and migrate into tissues. EMD is a negative prognostic factor of survival. Using flow cytometry and next-generation sequencing, we aimed to identify antigens and microRNAs (miRNAs) involved in EMD pathogenesis. Flow cytometry analysis revealed significant differences in the level of clonal plasma cells between MM and EMD patients, while the expression of CD markers was comparable between these two groups. Further, miR-26a-5p and miR-30e-5p were found to be significantly down-regulated in EMD compared to MM. Based on the expression of miR-26a-5p, we were able to distinguish these two groups of patients with high sensitivity and specificity. In addition, the involvement of deregulated miRNAs in cell cycle regulation, ubiquitin-mediated proteolysis and signaling pathways associated with infections or neurological disorders was observed using GO and KEGG pathways enrichment analysis. Subsequently, a correlation between the expression of analyzed miRNAs and the levels of CD molecules was observed. Finally, clinicopathological characteristics as well as CD antigens associated with the prognosis of MM and EMD patients were identified. Altogether, we identified several molecules possibly involved in the transformation of MM into EMD.

Genome-wide DNA methylation pattern in whole blood of patients with coal-burning arsenic poisoning

Exposure to coal-burning arsenic leads to an increased risk of cancer, multi-systems damage and chronic diseases, with DNA methylation one potential mechanism of arsenic toxicity. There are few studies on genome-wide methylation in the coal-burning arsenic poisoning population. Illumina 850 K methylation beadchip is the most suitable technology for DNA methylation of epigenome-wide association analysis. This study used 850 K to detect changes in Genome-wide DNA methylation in whole blood samples of 12 patients with coal-burning arsenic poisoning ( Arsenic poisoning group) and four healthy control participants (Healthy control group). There is clearly abnormal genome-wide DNA methylation in coal-burning arsenic poisoning, with 647 significantly different methylation positions, 524 different methylation regions and 335 significantly different methylation genes in arsenic poisoning patients compared with healthy controls. Further functional analysis of Gene ontology (GO) and Kyoto encyclopedia of genes (KEGG) found 592 GO items and 131 KEGG pathways between patients of coal-burning arsenic poisoning and healthy control. Then, analysis of gene degree and combined-score identified NAPRT1, NT5C3B, NEDD4L, SLC22A3 and RAB11B as target genes. Further validation by qRT-PCR indicates that mRNA expression of five genes changes significantly in the arsenic poisoning group (n = 72) compared to the healthy control group (n = 72). These results showed the genome-wide methylation pattern and highlighted five critical genes within the coal-burning arsenic poisoning population that involve Nicotinate and nicotinamide metabolism, Choline metabolism in cancer, and Ubiquitin mediated proteolysis. Next, the methylation profile of coal burning arsenic poisoning will be further excavation and the mechanism of coal burning arsenic poisoning will be further explored from five genes related pathways and functions.

Identification and validation of ubiquitin-proteasome system related genes as a prognostic signature for papillary renal cell carcinoma.

The Cancer Genome Atlas (TCGA) and GeneCards databases were utilized to find the clinical data and gene expression patterns of patients with PRCC. Univariate Cox regression analysis and absolute shrinkage and selection operator (LASSO) analyses identified a risk signature formed by ten optimal UPS genes. The predictive value of the risk signature in TCGA-PRCC cohorts was evaluated using Kaplan-Meier analysis and receiver operating characteristic (ROC) curves. By utilizing GO enrichment and the KEGG pathway, the interactions of differentially expressed genes connected to ubiquitin-mediated proteolysis were functionally examined. The protein expression of the hub genes was affirmed using the Human Protein Atlas (HPA) database. The effectiveness of particular CDC20 and UBE2C in vitro was confirmed by experimental research. Ten of the best ubiquitin-mediated proteolysis genes (UBE2C, DDB2, CBLC, BIRC3, PRKN, UBE2O, SIAH1, SKP2, UBC, and CDC20) were detected to create a risk signature. The high-risk score group stratified was associated with advanced tumor status and poor survival of PRCC patients. 10 genes were also found to be associated with the cell cycle pathway and ubiquitin-mediated proteolysis to GO and KEGG analysis. Of these 10 genes, CDC20 and UBE2C are highly expressed in tumor tissue and correlated with cancer immunity founded on the analyses of the expression of human protein atlas and TISIDB. The downregulation of UBE2C facilitated tumor inhibition and the anti-immune effect was confirmed by in vitro experiments. Our results indicate that the risk model created from the ubiquitin-mediated proteolysis genes can be reliably and accurately predict the prognosis of PRCC patients, highlighting its targeted value for PRCC treatment. Particularly, the expression of UBE2C may be crucial for the prognosis and immunological treatment of renal cancer.

Dynamic analysis of gene signatures in the progression of COPD.

Oxidative stress is an important amplifying mechanism in COPD; however, it is unclear how oxidative stress changes and what its exact amplification mechanism is in the pathological process. We aimed to dynamically analyse the progression of COPD and further elucidate the characteristics of each developmental stage and unveil the underlying mechanisms. We performed a holistic analysis by integrating Gene Expression Omnibus microarray datasets related to smoking, emphysema and Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification based on the concept of gene, environment and time (GET). Gene ontology (GO), protein-protein interaction (PPI) networks and gene set enrichment analysis (GSEA) were used to explore the changing characteristics and potential mechanisms. Lentivirus was used to promote HIF3A overexpression. In smokers versus nonsmokers, the GO term mainly enriched in "negative regulation of apoptotic process". In later transitions between stages, the main enriched terms were continuous progression of "oxidation-reduction process" and "cellular response to hydrogen peroxide". Logistic regression showed that these core differentially expressed genes (DEGs) had diagnostic accuracy in test (area under the curve (AUC)=0.828) and validation (AUC=0.750) sets. GSEA and PPI networks showed that one of the core DEGs, HIF3A, strongly interacted with the ubiquitin-mediated proteolysis pathway. Overexpression of HIF3A restored superoxide dismutase levels and alleviated the reactive oxygen species accumulation caused by cigarette smoke extract treatment. Oxidative stress was continuously intensified from mild emphysema to GOLD 4; thus, special attention should be paid to the identification of emphysema. Furthermore, the downregulated HIF3A may play an important role in the intensified oxidative stress in COPD.

Differential Expression and Bioinformatics Analysis of Plasma-Derived Exosomal circRNA in Type 1 Diabetes Mellitus.

Both exosome and circular RNA (circRNA) have been reported to participate in the pathogenesis of type 1 diabetes mellitus (T1DM). However, the exact role of exosomal circRNA in T1DM is largely unknown. Here, we identified the exosomal circRNA expression profiles in the plasma of T1DM patients and explored their potential function using bioinformatics analysis. Material and Methods. Exosomes were extracted by the size exclusion chromatography method from plasma of 10 T1DM patients and 10 age- and sex- matched control subjects. Illumina Novaseq6000 platform was used to detect the exosomal circRNA expression profiles. Multiple bioinformatics analysis was applied to investigate the potential biological functions of exosomal circRNAs. A total of 784 differentially expressed exosomal circRNAs have been identified in T1DM patients, of which 528 were upregulated and 256 were downregulated. Gene Ontology analysis enriched terms such as protein ubiquitination involved in ubiquitin-dependent protein catabolic protein (GO:0042787), membrane (GO:0016020), and GTPase activator activity (GO:0005096). The most enriched pathway in Kyoto Encyclopedia of Genes and Genomes was ubiquitin-mediated proteolysis (ko04120). The miRNA-targeting prediction method was used to identify the miRNAs that bind to circRNAs, and circRNA-miRNA-mRNA pathways were constructed, indicating that interactions between circRNA, miRNA, and gene might be involved in the disease progression. The present study identified the exosomal circRNA expression profiles in T1DM for the first time. Our results threw novel insights into the molecular mechanisms of T1DM.

DNMT2/TRDMT1 gene knockout compromises doxorubicin-induced unfolded protein response and sensitizes cancer cells to ER stress-induced apoptosis

The acidic, hypoxic and nutrient-deprived tumor microenvironment may induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) may exert an important cytoprotective role by promoting folding of newly synthesized proteins and cancer cell survival. The lack of DNMT2/TRDMT1 methyltransferase-mediated C38 tRNA methylation compromises translational fidelity that may result in the accumulation of misfolded and aggregated proteins leading to proteotoxic stress-related cell death. In the present study, DNMT2/TRDMT1 gene knockout-mediated effects were investigated during doxorubicin (DOX)-induced ER stress and PERK-, IRE1- and ATF6-orchestrated UPR in four genetically different cellular models of cancer (breast and cervical cancer, osteosarcoma and glioblastoma cells). Upon DOX stimulation, DNMT2/TRDMT1 gene knockout impaired PERK activation and modulated NSUN and 5-methylcytosine RNA-based responses and microRNA profiles. The lack of DNMT2/TRDMT1 gene in DOX-treated four cancer cell lines resulted in decreased levels of four microRNAs, namely, miR-23a-3p, miR-93-5p, miR-125a-5p and miR-191-5p involved in the regulation of several pathways such as ubiquitin-mediated proteolysis, amino acid degradation and translational misregulation in cancer. We conclude that DNMT2/TRDMT1 gene knockout, at least in selected cellular cancer models, affects adaptive responses associated with protein homeostasis networks that during prolonged ER stress may result in increased sensitivity to apoptotic cell death.

Whole-Genome Sequencing and Transcriptome Analysis of Ganoderma lucidum Strain Yw-1-5 Provides New Insights into the Enhanced Effect of Tween80 on Exopolysaccharide Production.

Ganoderma lucidum is an important medicinal mushroom widely cultured in Asian countries. Exopolysaccharides are bioactive compounds of G. lucidum with health benefits. Limited exopolysaccharide content hinders its extraction from G. lucidum. The addition of Tween80 had an enhanced effect on G. lucidum exopolysaccharide production in submerged fermentation. However, the mechanism of this effect remains unclear. In this study, we report on a high-quality assembly of G. lucidum strain yw-1-5 to lay the foundation for further transcriptome analysis. The genome sequence was 58.16 Mb and consisted of 58 scaffolds with an N50 of 4.78 Mb. A total of 13,957 protein-coding genes were annotated and Hi-C data mapped to 12 pseudo-chromosomes. Genes encoding glycosyltransferases and glycoside hydrolases were also obtained. Furthermore, RNA-seq was performed in a Tween80-treated group and control group for revealing the enhanced effect of Tween80 on exopolysaccharide production. In total, 655 genes were identified as differentially expressed, including 341 up-regulated and 314 down-regulated. Further analysis of differentially expressed genes showed that groups of MAPK, amino sugar and nucleotide sugar metabolism, autophagy, ubiquitin-mediated proteolysis, peroxisome, starch and sucrose metabolism, TCA cycle, glycolysis/gluconeogenesis KEGG pathway, glycosyltransferases and glycoside hydrolases played important roles in the enhanced effect of Tween80 on exopolysaccharide production. This work provides a valuable resource for facilitating our understanding of the synthesis of polysaccharides and accelerating the breeding of new strains with a high content of exopolysaccharides.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Serum extracellular vesicle MicroRNAs as candidate biomarkers for acute rejection in patients subjected to liver transplant.

Acute rejection (AR) is a common and grave complication of liver transplantation (LT). The diagnosis of AR is challenging because it has nonspecific clinical features and requires invasive procedures. Since extracellular vesicles (EVs) are promising candidates as indicators for diagnosis of various diseases, this study aimed to identify serum EV microRNAs (miRNAs) as potential biomarkers for AR in patients subjected to LT. We collected clinical information and serum samples from the liver transplant recipients with and without AR (non-AR). EVs from the serum were isolated via ultracentrifugation and identified using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. EV RNA was extracted and sequenced on an Illumina HiSeq 2500/2000 platform to identify differentially expressed miRNAs between the groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the target gene candidates of the differentially expressed miRNAs to test their functions in biological systems. Then, we validated 12 differentially expressed miRNAs by quantitative real-time PCR. The results demonstrated that 614 EV miRNAs were significantly altered (387 up regulated and 227 down regulated) between non-AR and AR patients. GO enrichment analysis revealed that these target genes were related to cellular processes, single-organism processes, biological regulation, metabolic processes, cells, cell parts, protein-binding processes, nucleoid binding, and catalytic activity. Furthermore, KEGG pathway analysis demonstrated that the target genes of the differentially expressed miRNAs were primarily involved in ubiquitin-mediated proteolysis, lysosomes, and protein processing in the endoplasmic reticulum. miR-223 and let-7e-5p in AR patients were significantly up-regulated compared to those in non-AR patients, whereas miR-199a-3p was significantly down-regulated, which was consistent with sequencing results. The expression of serum EV miRNAs (up-regulated: miR-223 and let-7e-5p and miR-486-3p; down regulated: miR-199a-3p, miR-148a-3p and miR-152-3p) in AR patients was significantly different from that in non-AR patients, and these miRNAs can serve as promising diagnostic biomarkers for AR in patients subjected to liver transplant.

Identification and verification of the prognostic value of CUL7 in colon adenocarcinoma.

CUL7, a gene composed of 26 exons associated with cullin 7 protein, is also an E3 ligase that is closely related to cell senescence, apoptosis, and cell transformation and also plays an important role in human cancer. However, there is no systematic pan-cancer analysis has been performed to explore its role in prognosis and immune prediction. In this study, the expression of CUL7 in colon adenocarcinoma (COAD) was investigated to determine its prognosis value. First, based on the Cancer Genome Atlas (TCGA), Genotypic-Tissue Expression Project(GTEx), Cancer Cell Line Encyclopedias(CCLE), and TISIDB database, the potential role of CUL7 in different tumors was explored. Subsequently, the expression of CUL7 in COAD was explored and verified by Immunohistochemistry (IHC). Furthermore, the mutation frequency of CUL7 in COAD was analyzed, and the prognostic value of CUL7 in COAD was discussed. In addition, the nomogram was constructed, and its prognostic value was verified by follow-up data from Jiangmen Central Hospital. Finally, PPI network analysis explored the potential biological function of CUL7 in COAD. The results show that CUL7 is upregulated in most tumors, which is significantly associated with poor survival. At the same time, CUL7 is correlated with the clinical stage and immune landscape of various tumors. In colorectal cancer, CUL7 was overexpressed in tumor tissues by IHC with a mutation frequency of about 4%. CUL7 is an independent prognostic factor for colorectal cancer. The nomogram constructed has effective predictive performance, and external databases proved the prognostic value of CUL7. In addition, PPI network analysis showed that CUL7 was closely related to FBXW8, and further pathway enrichment analysis showed that CUL7 was mainly involved in ubiquitin-mediated proteolysis. Therefore, our study provides a comprehensive understanding of the potential role of CUL7 in different tumors, and CUL7 might be a prognostic marker for COAD.

Expression Profiles of Differentially Expressed Circular RNAs and circRNA-miRNA-mRNA Regulatory Networks in SH-SY5Y Cells Infected with Coxsackievirus B5.

Coxsackievirus B5 (CVB5) is the causative agent of hand, foot, and mouth disease (HFMD) that can cause neurological complications and fatalities. Circular RNA (circRNA) has been shown to play an important role in regulating pathogenic processes. However, the functions of circRNA in response to CVB5 infection remain unclear. In our research, RNA-seq was employed to analyze the expression profiles of circRNAs in SH-SY5Y cells with or without CVB5 infection. Out of 5,665 circRNAs identified to be expressed in SH-SY5Y cells, 163 circRNAs were found to be differentially expressed significantly. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the differentially expressed circRNAs were mainly involved in ubiquitin-mediated proteolysis and signaling pathways during CVB5 infection. Additionally, RT-qPCR was used to validate the RNA-seq data, and a circRNA–miRNA–mRNA interaction network was constructed based on two circRNAs, such as hsa_circ_0008378 and novel_circ_0014617, which were associated with the regulation of innate immune response in host cells. Additionally, we confirmed the two circRANs up-regulated the key factors in the IFN-I signaling pathway, hampering viral replication. Our data provide a new perspective that facilitates further understanding of the virus-host mechanism.

Fine-Tuned Immune Antagonism and Nodule-Specific Cysteine-Rich Peptides Govern the Symbiotic Specificity Between Alfalfa Cultivars and Ensifer meliloti

Alfalfa expresses significantly distinct sets of genes in response to infection with different rhizobial strains at the below species level (i.e., biotype or strain). However, differences in the transcriptomic profiles of two alfalfa cultivars nodulated by a single rhizobium strain have been largely unexamined. In this study, comparative RNA-seq analysis of two alfalfa cultivars, Medicago sativa cv. Gannong No. 3 (G3) and cv. Gannong No. 9 (G9) inoculated with one Ensifer meliloti strain LL2, with varying symbiotic performance, was conducted, followed by hub gene interaction network construction based on weighted gene co-expression network analysis (WGCNA). The G9-LL2 symbiotic system showed better nodule formation, nitrogen fixation, and growth characteristics than the G3-LL2 system. Compared with the non-inoculated control, the LL2-inoculated G9 plants (10,053) produced more differentially expressed genes (DEGs) than the LL2-inoculated G3 plants (7112). A group of 227 genes displayed completely distinguished expression in G9 (6.63 < log2(FC) < 15.45) and G3 (‒ 3.05 < log2(FC) < 12.05), which are primarily involved in encoding nodule-specific cysteine-rich peptides (NCRs), nodulin, and leghemoglobin. Although genes with predicted roles in nitrogen metabolism were primarily upregulated and almost all of those in ubiquitin-mediated proteolysis and plant–pathogen interaction were suppressed, interestingly, a consistently higher expression level measured by log2(FC) was observed in G9 plants. Hub gene interaction networks showed that NCRs, late nodulin, and genes related to plant immunity (TIR-NBS-LRR, defensin, thioredoxin, thionine, and polygalacturonase) regulate other genes at the source node positions. After successful initiation of nodulation in both alfalfa cultivars G3 and G9 by E. meliloti strain LL2, G9 achieved preferable outcomes of rhizobia–alfalfa symbiosis by equilibrating the antagonism and compatibility of plant immunity. It elevated PTI, suppressed defense and ETI, and enhanced nitrogen fixation and utilization efficiency by inducing the expression of genes encoding NIN, NFH1, LysM-RLK, LRP, NCRs, nodulin, and leghemoglobin. Hub genes were predominantly associated with highly specific rhizobia–alfalfa symbiosis positively governed by NCRs and fine-tuned immune antagonism, comprising NCRs, late nodulin, and TIR-NBS-LRR. These findings provide insights into the genetic mechanisms underlying the modification and efficient utilization of semi-compatible and incompatible rhizobial resources.