Differentially Expressed mRNAs Research Articles

Comparative Transcriptomic Analysis of mRNAs, miRNAs and lncRNAs in the Longissimus dorsi Muscles between Fat-Type and Lean-Type Pigs.

In pigs, meat quality and production are two important traits affecting the pig industry and human health. Compared to lean-type pigs, fat-type pigs contain higher intramuscular fat (IMF) contents, better taste and nutritional value. To uncover genetic factors controlling differences related to IMF in pig muscle, we performed RNA-seq analysis on the transcriptomes of the Longissimus dorsi (LD) muscle of Laiwu pigs (LW, fat-type pigs) and commercial Duroc × Landrace × Yorkshire pigs (DLY, lean-type pigs) at 150 d to compare the expression profiles of mRNA, miRNA and lncRNA. A total of 225 mRNAs, 12 miRNAs and 57 lncRNAs were found to be differentially expressed at the criteria of |log2(foldchange)| > 1 and q < 0.05. The mRNA expression of LDHB was significantly higher in the LD muscle of LW compared to DLY pigs with log2(foldchange) being 9.66. Using protein interaction prediction method, we identified more interactions of estrogen-related receptor alpha (ESRRA) associated with upregulated mRNAs, whereas versican (VCAN) and proenkephalin (PENK) were associated with downregulated mRNAs in LW pigs. Integrated analysis on differentially expressed (DE) mRNAs and miRNAs in the LD muscle between LW and DLY pigs revealed two network modules: between five upregulated mRNA genes (GALNT15, FKBP5, PPARGC1A, LOC110258214 and LOC110258215) and six downregulated miRNA genes (ssc-let-7a, ssc-miR190-3p, ssc-miR356-5p, ssc-miR573-5p, ssc-miR204-5p and ssc-miR-10383), and between three downregulated DE mRNA genes (IFRD1, LOC110258600 and LOC102158401) and six upregulated DE miRNA genes (ssc-miR1379-3p, ssc-miR1379-5p, ssc-miR397-5p, ssc-miR1358-5p, ssc-miR299-5p and ssc-miR1156-5p) in LW pigs. Based on the mRNA and ncRNA binding site targeting database, we constructed a regulatory network with miRNA as the center and mRNA and lncRNA as the target genes, including GALNT15/ssc-let-7a/LOC100523888, IFRD1/ssc-miR1379-5p/CD99, etc., forming a ceRNA network in the LD muscles that are differentially expressed between LW and DLY pigs. Collectively, these data may provide resources for further investigation of molecular mechanisms underlying differences in meat traits between lean- and fat-type pigs.

Elucidation of endothelial progenitor cell dysfunction in diabetes by RNA sequencing and constructing lncRNA-miRNA-mRNA competing endogenous RNA network.

With the rapid increase in the incidence of diabetes, non-healing diabetic wounds have posed a huge challenge to public health. Endothelial progenitor cell (EPC) has been widely reported to promote wound repairing, while its number and function were suppressed in diabetes. However, the specific mechanisms and competing endogenous RNA (ceRNA) network of EPCs in diabetes remain largely unknown. Thus, the transcriptome analyses were carried in the present study to clarify the mechanism underlying EPCs dysfunction in diabetes. EPCs were successfully isolated from rats. Compared to the control, diabetic rat-derived EPCs displayed impaired proliferation, migration, and tube formation ability. The differentially expressed (DE) RNAs were successfully identified by RNA sequencing in the control and diabetic groups. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that DE mRNAs were significantly enriched in terms and pathways involved in the functions of EPCs and wound healing. Protein-protein interaction networks revealed critical DE mRNAs in the above groups. Moreover, the whole lncRNA-miRNA-mRNA ceRNA network was constructed, in which 9 lncRNAs, 9 mRNAs, and 5 miRNAs were further validated by quantitative real-time polymerase chain reaction. Rno-miR-10b-5p and Tgfb2 were identified as key regulators of EPCs dysfunction in diabetes. The present research provided novel insight into the underlying mechanism of EPCs dysfunction in diabetes and prompted potential targets to restore the impaired functions, thus accelerating diabetic wound healing. KEY MESSAGES: • Compared to the control, diabetic rat-derived EPCs displayed impaired proliferation, migration, and tube formation ability. • The DE RNAs were successfully identified by RNA sequencing in the control and diabetic groups and analyzed by DE, GO, and KEGG analysis. • PPI and lncRNA-miRNA-mRNA ceRNA networks were constructed. • 9 lncRNAs, 9 mRNAs, and 5 miRNAs were further validated by qRT-PCR. • Rno-miR-10b-5p and Tgfb2 were identified as key regulators of EPCs dysfunction in diabetes.

RNA sequencing uncover crucial genes mediating progression of large-artery atherosclerotic and small-artery occlusion ischemic stroke

PurposeThe goal of our study is to uncover the pathogenesis of large-artery atherosclerotic ischemic stroke (LAAIS) and small-artery occlusion ischemic stroke (SAOIS) and analyze their difference using RNA sequencing. MethodsRNA sequencing was used to filtrate differentially expressed mRNAs (DEmRNAs) and differentially expressed lncRNAs (DElncRNAs) in LAAIS and SAOIS. Specific DEmRNAs and DElncRNAs in LAAIS and SAOIS were further found. Functional annotation and DElncRNA-DEmRNA co-expression network were built to reveal biological function of DEmRNAs. ResultsA total of 832 DEmRNAs and 96 DElncRNAs were identified in LAAIS vs normal controls. 587 DEmRNAs and 105 DElncRNAs were identified in SAOIS vs normal controls. In LAAIS vs SAOIS, 636 DEmRNAs and 112 DElncRNAs were identified. Among which, 571 DEmRNAs and 61 DElncRNAs were LAAIS specific DEmRNAs and DElncRNAs, respectively. 325 DEmRNAs and 66 DElncRNAs were respectively SAOIS specific DEmRNAs and DElncRNAs. We also obtained 3086 LAAIS specific DElncRNA-DEmRNA co-expression pairs and 661 SAOIS specific DElncRNA-DEmRNA co-expression pairs. Oxidative phosphorylation and Alzheimer's disease were significantly enriched pathways in both LAAIS specific DEmRNAs and DEmRNAs in LAAIS specific DElncRNA-DEmRNA co-expression network. ECM-receptor interaction, hypertrophic cardiomyopathy and dilated cardiomyopathy were significantly enriched pathways in both SAOIS specific DEmRNAs and DEmRNAs in SAOIS specific DElncRNA-DEmRNA co-expression network. ConclusionThis finding may help to understand the mechanisms of LAAIS and SAOIS and offer novel clues for finding specific biomarkers for LAAIS and SAOIS.

Protective effects and regulatory pathways of melatonin in traumatic brain injury mice model: Transcriptomics and bioinformatics analysis.

Traumatic brain injury (TBI) is the leading cause of disability and mortality globally. Melatonin (Mel) is a neuroendocrine hormone synthesized from the pineal gland that protects against TBI. Yet, the precise mechanism of action is not fully understood. In this study, we examined the protective effect and regulatory pathways of melatonin in the TBI mice model using transcriptomics and bioinformatics analysis. The expression profiles of mRNA, long non-coding RNA (lncRNA), microRNA (miRNA), and circular RNA (circRNA) were constructed using the whole transcriptomes sequencing technique. In total, 93 differentially expressed (DE) mRNAs (DEmRNAs), 48 lncRNAs (DElncRNAs), 59 miRNAs (DEmiRNAs), and 59 circRNAs (DEcircRNAs) were identified by the TBI mice with Mel treatment compared to the group without drug intervention. The randomly selected coding RNAs and non-coding RNAs (ncRNAs) were identified by quantitative real-time polymerase chain reaction (qRT-PCR). To further detect the biological functions and potential pathways of those differentially expressed RNAs, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were executed. In our research, the regulatory network was constructed to show the relationship of lncRNA-RBPs. The lncRNA-mRNA co-expression network was established based on the Pearson coefficient to indicate the expression correlations. Moreover, the DEcircRNA–DEmiRNA–DEmRNA and DElncRNA–DEmiRNA–DEmRNA regulatory networks were constructed to demonstrate the regulatory relationship between ncRNAs and mRNA. Finally, to further verify our predicted results, cytoHubba was used to find the hub gene in the synaptic vesicle cycle pathway, and the expression level of SNAP-25 and VAMP-2 after melatonin treatment were detected by Western blotting and immunofluorescence. To sum up, these data offer a new insight regarding the molecular effect of melatonin treatment after TBI and suggest that the high-throughput sequencing and analysis of transcriptomes are useful for studying the drug mechanisms in treatment after TBI.

Characterization of differentially expressed and lipid metabolism-related lncRNA-mRNA interaction networks during the growth of liver tissue through rabbit models.

BackgroundCharacterization the long non-coding RNAs (lncRNAs) and their regulated mRNAs involved in lipid metabolism during liver growth and development is of great value for discovering new genomic biomarkers and therapeutic targets for fatty liver and metabolic syndrome.Materials and methodsLiver samples from sixteen rabbit models during the four growth stages (birth, weaning, sexual maturity, and somatic maturity) were used for RNA-seq and subsequent bioinformatics analyses. Differentially expressed (DE) lncRNAs and mRNAs were screened, and the cis/trans-regulation target mRNAs of DE lncRNAs were predicted. Then the function enrichment analyses of target mRNAs were performed through Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. The target protein interaction (PPI) and lncRNA-mRNA co-expression networks were constructed using string version 11.0 platform and R Stats. Finally, six lncRNAs and six mRNAs were verified taking RT-qPCR.ResultsLiver Oil Red O detection found that the liver showed time-dependent accumulation of lipid droplets. 41,095 lncRNAs, 30,744 mRNAs, and amount to 3,384 DE lncRNAs and 2980 DE mRNAs were identified from 16 cDNA sequencing libraries during the growth of liver. 689 out of all DE lncRNAs corresponded to 440 DE mRNAs by cis-regulation and all DE mRNAs could be regulated by DE lncRNAs by trans-regulation. GO enrichment analysis showed significant enrichment of 892 GO terms, such as protein binding, cytosol, extracellular exsome, nucleoplasm, and oxidation-reduction process. Besides, 52 KEGG pathways were significantly enriched, including 11 pathways of lipid metabolism were found, like Arachidonic acid metabolism, PPAR signaling pathway and Biosynthesis of unsaturated fatty acids. After the low expression DE mRNAs and lncRNAs were excluded, we further obtained the 54 mRNAs were regulated by 249 lncRNAs. 351 interaction pairs were produced among 38 mRNAs and 215 lncRNAs through the co-expression analysis. The PPI network analysis found that 10 mRNAs such as 3β-Hydroxysteroid-Δ24 Reductase (DHCR24), lathosterol 5-desaturase (SC5D), and acetyl-CoA synthetase 2 (ACSS2) were highly interconnected hub protein-coding genes. Except for MSTRG.43041.1, the expression levels of the 11 genes by RT-qPCR were the similar trends to the RNA-seq results.ConclusionThe study revealed lncRNA-mRNA interation networks that regulate lipid metabolism during liver growth, providing potential research targets for the prophylaxis and treatment of related diseases caused by liver lipid metabolism disorders.

Abstract 070: ONE UP-REGULATED NOVEL MICRORNA AND 4 DOWN-REGULATED MRNA TARGETS WERE IDENTIFIED IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF HYPERTENSIVE PATIENTS WITH METABOLIC SYNDROME

Introduction: Hypertension is associated with target organ damage such as kidney injury. The immune system plays a role in hypertension and target organ damage. Activation of T cells has been reported among peripheral blood mononuclear cells (PBMCs) of patients with HTN. MicroRNAs are crucial post-transcriptional regulators of immune cell development and function. Hypothesis: MicroRNAs play a role in the activation of immune cells in hypertension with target organ damage in humans. Methods: Normotensive subjects (NTN) and patients with hypertension (HTN) associated or not with at least 2 other features of the metabolic syndrome (MetS) or chronic kidney disease (CKD) were studied (n=15-16). PBMCs were isolated from blood, RNA extracted, small and total RNA sequencing (RNA-seq) using an Illumina HiSeq-2500 and data were analyzed using a systems biology approach. Differentially expressed (DE) microRNAs and mRNAs were identified with fold change (FC) &gt;2 and &gt;1.5, respectively, and P &lt;0.005. DE miRNAs with RNA-seq count number (CN) &gt;500, and predicted targets by TargetScan with CN&gt;300 were validated by reverse transcription-quantitative PCR (RT-qPCR) in PBMCs. Results: RNA-seq identified DE microRNAs and mRNAs in HTN (22 and 19), MetS (57 and 401) and CKD (6 and 26) compared to NTN. RT-qPCR validated a novel miRNA (miR-pl-86 [2-fold up]) and 4 predicted mRNA targets (WD repeat domain 89 [ WDR89 , 51% down], Dmx like 1 [ DMXL1 , 52% down], zinc finger protein 600 [ ZNF600 , 63% down] and NOC3 like DNA replication regulator [ NOC3L , 61% down]) in MetS vs NTN ( P &lt;0.05). RNA-seq results were correlated with RT-qPCR data for miR-pl-86 (R 2 =0.37, P &lt;5.5E-07, n=56) , WDR89 (R 2 =0.29, P &lt;1.4E-05, n=57) , DMXL1 (R 2 =0.33, P &lt;2.7E-06, n=57) , ZNF600 (R 2 =0.25, P &lt;7.9E-05, n=57) , and NOC3L (R 2 =0.27, P &lt;3.5E-05, n=57). Conclusion: This study identified one up-regulated novel microRNA and 4 down-regulated mRNA targets in PBMCs of patients with HTN and MetS.

Interactions Among Non-Coding RNAs and mRNAs in the Trigeminal Ganglion Associated with Neuropathic Pain.

BackgroundRecent studies have demonstrated the contribution of non-coding RNAs (ncRNAs) to neuropathic pain. However, the expression profile of ncRNAs in the trigeminal ganglion (TG) and their functional mechanism underlying trigeminal neuropathic pain are still unclear.MethodsIn the present study, the trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve (CCI-ION) was used to study the expression profile and potential regulatory mechanism of miRNAs, lncRNAs, circRNAs, and mRNAs in the TG by RNA-sequencing (RNA-seq) and bioinformatics analysis. CCI-ION mice suffered from mechanical allodynia from 3 days to 28 days after surgery.ResultsThe RNA-seq results discovered 67 miRNAs, 216 lncRNAs, 14 circRNAs, 595 mRNAs, and 421 genes were differentially expressed (DE) in the TG of CCI-ION mice 7 days after surgery. And 39 DEGs were known pain genes. Besides, 5 and 35 pain-related DE mRNAs could be targeted by 6 DE miRNAs and 107 DE lncRNAs, respectively. And 23 pain-related DEGs had protein–protein interactions (PPI) with each other. GO analysis indicated membrane-related cell components and binding-related molecular functions were significantly enriched. KEGG analysis showed that nociception-related signaling pathways were significantly enriched for DE ncRNAs and DEGs. Finally, the competing endogenous RNA (ceRNA) regulatory network of DE lncRNA/DE circRNA-DE miRNA-DE mRNA existed in the TG of mice with trigeminal neuropathic pain.ConclusionOur findings demonstrate ncRNAs are involved in the development of trigeminal neuropathic pain, possibly through the ceRNA mechanism, which brings a new bright into the study of trigeminal neuropathic pain and the development of novel treatments targeting ncRNAs.

Comprehensive Analysis of Serum Small Extracellular Vesicles-Derived Coding and Non-Coding RNAs from Retinoblastoma Patients for Identifying Regulatory Interactions.

Simple SummaryThe diagnosis of retinoblastoma (RB) is usually made by clinical examination and imaging modalities. Routine tissue biopsy is not done due to the risk of extraocular spread. Blood-based RNA cargoes could be promising surrogate markers for RB diagnosis and prognostication. Our data indicated that the size, morphology, and zeta potential (ZP) of RB and non-RB serum extracellular vesicles (EVs) met standard exosome properties with similar concentrations. MALTA1, AFAP1-AS1, miR-145, and miR-101 were identified as hub non-coding RNAs that promote RB progression by targeting cyclins, cyclin-dependent kinases, c-MYC, EZH2, ZEB1, TP53, and BCL2. Along with these, the aberrantly expressed miRNAs, lncRNAs, and their target mRNAs of RB EVs were implicated in cell cycle, metabolism, and tumor-associated signaling pathways. The differential expression of EV RNAs in RB compared to controls may aid in the identification of possible serum prognostic biomarkers for RB.The present study employed nanoparticle tracking analysis, transmission electron microscopy, immunoblotting, RNA sequencing, and quantitative real-time PCR validation to characterize serum-derived small extracellular vesicles (sEVs) from RB patients and age-matched controls. Bioinformatics methods were used to analyze functions, and regulatory interactions between coding and non-coding (nc) sEVs RNAs. The results revealed that the isolated sEVs are round-shaped with a size < 150 nm, 5.3 × 1011 ± 8.1 particles/mL, and zeta potential of 11.1 to −15.8 mV, and expressed exosome markers CD9, CD81, and TSG101. A total of 6514 differentially expressed (DE) mRNAs, 123 DE miRNAs, and 3634 DE lncRNAs were detected. Both miRNA-mRNA and lncRNA-miRNA-mRNA network analysis revealed that the cell cycle-specific genes including CDKNI1A, CCND1, c-MYC, and HIF1A are regulated by hub ncRNAs MALAT1, AFAP1-AS1, miR145, 101, and 16-5p. Protein-protein interaction network analysis showed that eye-related DE mRNAs are involved in rod cell differentiation, cone cell development, and retinol metabolism. In conclusion, our study provides a comprehensive overview of the RB sEV RNAs and regulatory interactions between them.

Bioinformatics analysis of the circRNA-miRNA-mRNA network for atrial fibrillation.

Atrial fibrillation (AF) is a chronic and progressive disease, with advancing age, the morbidity of which will increase exponentially. Circular ribonucleic acids (RNAs; circRNAs) have gained a growing attention in the development of AF in recent years. The purpose of this study is to explore the mechanism of circRNA regulation in AF, in particular, the intricate interactions among circRNA, microRNA (miRNA), and messenger RNA (mRNA). Three datasets (GSE129409, GSE68475, and GSE79768) were obtained from the Gene Expression Omnibus database to screen differentially expressed (DE) circRNAs, DE miRNAs, and DE mRNAs in AF, respectively. Based on circRNA-miRNA pairs and miRNA-mRNA pairs, a competing endogenous RNAs (ceRNAs) network was built. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis of DE mRNAs in the network were performed and protein-protein interaction (PPI) networks were established to identify hub genes. Finally, a circRNA-miRNA-hub gene subnetwork was constructed. A total of 103 DE circRNAs, 16 DE miRNAs, and 110 DE mRNAs were screened in AF. Next, ceRNAs network in AF was constructed with 3 upregulated circRNAs, 2 downregulated circRNAs, 2 upregulated miRNAs, 2 downregulated miRNAs, 17 upregulated mRNAs, and 24 downregulated mRNAs. Thirty GO terms and 6 KEGG pathways were obtained. Besides, 6 hub genes (C-X-C chemokine receptor type 4 [CXCR4], C-X-C chemokine receptor type 2 [CXCR2], C-X-C motif chemokine 11 [CXCL11], neuromedin-U, B1 bradykinin receptor, and complement C3) were screened from constructing a PPI network. Finally, a circRNA-miRNA-hub gene subnetwork with 10 regulatory axes was constructed to describe the interactions among the differential circRNAs, miRNA, and hub genes. We speculated that hsa_circRNA_0056281/hsa_circRNA_0006665 -hsa-miR-613-CXCR4/CXCR2/CXCL11 regulatory axes and hsa_circRNA_0003638-hsa-miR-1207-3p-CXCR4 regulatory axis may be associated with the pathogenesis of AF.

Early-Life Exposure of Pigs to Topsoil Alters miRNA and mRNA Expression in Peripheral Blood Mononuclear Cells.

Exposure to less-hygienic conditions during early childhood has been associated with stimulation and development of the immune system. A recent study indicated that exposure of piglets to soil-borne microbes during lactation was related with modulation of gut microbiota and immune function. To identify the potential molecular mechanisms and pathways impacted by early-life topsoil exposure, we analyzed the messenger RNA (mRNA) and micro-RNA (miRNA) expression in peripheral blood mononuclear cells (PBMCs) from these piglets. Total RNA was extracted from the PBMCs of piglets exposed to topsoil only from d 4–d 21 of life (mRNA n = 6; miRNA n = 5) or unexposed control pigs (mRNA n = 6; miRNA n = 8) at 11, 20, and 56 days of age. Small RNA and mRNA were sequenced with 50-bp single-end reads using Illumina chemistry. Sequence data were quality checked with FASTQC software and aligned to the Sscrofa 11.1 genome with the STAR aligner for mRNA and mirDeep2 for miRNA. Differential expression (DE) analysis was performed using PROC Glimmix of SAS to evaluate changes in expression due to topsoil exposure over time with genes declared DE at a false discovery rate (FDR) of q < 0.10. A total of 138 mRNA and 21 miRNAs were identified as DE for the treatment by age interaction. Ontology enrichment analysis of DE mRNA revealed Gene ontology (GO) terms directly involved in the connection between T-cell and antigen-presenting cells that are associated with T-cell activation. Key regulatory genes identified include PTPRJ, ITGB3, TRBV30, CD3D, mir-143, mir-29, and mir-148a. While these results require validation, this study provides data supporting the hypothesis that less-hygienic environments during early life may contribute to the development of the immune system.

MRNA and lncRNA expression profiles of liver tissues in children with biliary atresia

Progressive liver fibrosis is the most common phenotype in biliary atresia (BA). A number of pathways contribute to the fibrosis process so comprehensive understanding the mechanisms of liver fibrosis in BA will pave the way to improve patient's outcome after operation. In this study, the differentially expressed profiles of mRNAs and long non-coding RNAs from BA and choledochal cyst (CC) liver tissues were investigated and analyzed, which may provide potential clues to clarify hepatofibrosis mechanism in BA. A total of two BA and two CC liver tissue specimens were collected, the expression level of mRNAs and lncRNAs was detected by RNA sequencing. Differentially expressed mRNAs (DEmRNAs) were functionally annotated and protein-protein interaction networks (PPI) was established to predict the biological roles and interactive relationships. Differentially expressed lncRNAs (DElncRNAs) nearby targeted DEmRNA network and DElncRNA-DEmRNA co-expression network were constructed to further explore the roles of DElncRNAs in BA pathogenesis. The expression profiles of significant DEmRNAs were validated in Gene Expression Omnibus database. A total of 2,086 DEmRNAs and 184 DElncRNAs between BA and CC liver tissues were obtained. DEmRNAs were enriched in 521 Gene Ontology terms and 71 Kyoto Encyclopedia of Genes and Genomes terms which were mainly biological processes and metabolic pathways related to immune response and inflammatory response. A total of five hub proteins (TYRO protein tyrosine kinase binding protein, C-X-C motif chemokine ligand 8, pleckstrin, Toll-like receptor 8 and C-C motif chemokine receptor 5) were found in the PPI networks. A total of 31 DElncRNA-nearby-targeted DEmRNA pairs and 2,337 DElncRNA-DEmRNA co-expression pairs were obtained. The expression of DEmRNAs obtained from RNA sequencing were verified in GSE46960 dataset, generally. The present study identified key genes and lncRNAs participated in BA associated liver fibrosis, which may present a new avenue for understanding the patho-mechanism for hepatic fibrosis in BA.

Construction, bioinformatics analysis, and validation of competitive endogenous RNA networks in ulcerative colitis.

Background: Ulcerative colitis (UC) is a common chronic disease of the digestive system. Recently, competitive endogenous RNAs (ceRNAs) have been increasingly used to reveal key mechanisms for the pathogenesis and treatment of UC. However, the role of ceRNA in UC pathogenesis has not been fully clarified. This study aimed to explore the mechanism of the lncRNA-miRNA-mRNA ceRNA network in UC and identify potential biomarkers and therapeutic targets. Materials and Methods: An integrative analysis of mRNA, microRNA (miRNA), and long non-coding RNA (lncRNA) files downloaded from the Gene Expression Omnibus (GEO) was performed. Differentially expressed mRNA (DE-mRNAs), miRNA (DE-miRNAs), and lncRNA (DE-lncRNAs) were investigated between the normal and UC groups by the limma package. A weighted correlation network analysis (WGCNA) was used to identify the relative model for constructing the ceRNA network, and, concurrently, miRWalk and DIANA-LncBase databases were used for target prediction. Consecutively, the Gene Ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) pathway, and Reactome pathway enrichment analyses, protein-protein interaction (PPI) network, Cytohubba, and ClueGO were performed to identify hub genes. Additionally, we examined the immune infiltration characteristics of UC and the correlation between hub genes and immune cells using the immuCellAI database. Finally, the expression of potential biomarkers of ceRNA was validated via qRT-PCR in an experimental UC model induced by dextran sulfate sodium (DSS). Result: The ceRNA network was constructed by combining four mRNAs, two miRNAs, and two lncRNAs, and the receiver operating characteristic (ROC) analysis showed that two mRNAs (CTLA4 and STAT1) had high diagnostic accuracy (area under the curve [AUC] > 0.9). Furthermore, CTLA4 up-regulation was positively correlated with the infiltration of immune cells. Finally, as a result of this DSS-induced experimental UC model, CTLA4, MIAT, and several associate genes expression were consistent with the results of previous bioinformatics analysis, which proved our hypothesis. Conclusion: The investigation of the ceRNA network in this study could provide insight into UC pathogenesis. CTLA4, which has immune-related properties, can be a potential biomarker in UC, and MIAT/miR-422a/CTLA4 ceRNA networks may play important roles in UC.

Integrated microarray for identifying the hub mRNAs and constructed miRNA-mRNA network in coronary in-stent restenosis.

As a major complication after percutaneous coronary intervention (PCI) in patients who suffer from coronary artery disease, in-stent restenosis (ISR) poses a significant challenge for clinical management. A miRNA-mRNA regulatory network of ISR can be constructed to better reveal the occurrence of ISR. The relevant data set from the Gene Expression Omnibus (GEO) database was downloaded, and 284 differentially expressed miRNAs (DE-miRNAs) and 849 differentially expressed mRNAs (DE-mRNAs) were identified. As predicted by online tools, 65 final functional genes (FmRNAs) were overlapping DE-mRNAs and DE-miRNAs target genes. In the biological process (BP) terms of gene ontology (GO) functional analysis, the FmRNAs were mainly enriched in the cellular response to peptide, epithelial cell proliferation, and response to peptide hormone. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the FmRNAs were mainly enriched in breast cancer, endocrine resistance, and Cushing syndrome. Jun proto-oncogene, activator protein-1 (AP-1) transcription factor subunit (JUN), insulin-like growth factor 1 receptor (IGF1R), member RAS oncogene family (RAB14), specificity protein 1 (SP1), protein tyrosine phosphatase nonreceptor type 1 (PTPN1), DDB1 and CUL4 associated factor 10 (DCAF10), retinoblastoma-binding protein 5 (RBBP5), and eukaryotic initiation factor 4A-I (EIF4A1) were hub genes in the protein-protein interaction network (PPI network). The miRNA-mRNA network containing DE-miRNAs and hub genes was built. Hsa-miR-139-5p-JUN, hsa-miR-324-5p-SP1 axis pairs were found in the miRNA-mRNA network, which could promote ISR development. The aforementioned results indicate that the miRNA-mRNA network constructed in ISR has a regulatory role in the development of ISR and may provide new approaches for clinical treatment and experimental development.

Long Noncoding RNA LEMD1-AS1 Increases LEMD1 Expression and Activates PI3K-AKT Pathway to Promote Metastasis in Oral Squamous Cell Carcinoma.

Background The survival rate of oral squamous cell carcinoma (OSCC) is only 50% due to a high incidence of metastasis. Long noncoding RNAs (lncRNAs) play a crucial role in OSCC genesis and progression, although their potential role in the metastasis of OSCC remains unclear. Methods The transcriptome of 5 metastatic and 5 nonmetastatic OSCC samples were assessed by RNA sequencing. The biological functions and regulatory mechanisms of LEMD1-AS1 in OSCC were explored by in vitro and in vivo assays. Results We identified 487 differentially expressed mRNAs (DEmRNAs) and 1507 differentially expressed lncRNAs (DElncRNAs) in OSCC with cervical lymph node (LN) metastasis relative to the nonmetastatic samples. In addition, both LEMD1-AS1 and its cognate LEMD1 were up-regulated in metastatic OSCC compared to nonmetastatic OSCC. Gain-of-function, loss-of-function, and rescue experiments indicated that LEMD1-AS1 upregulated LEMD1 to increase OSCC migration and invasion in vitro and in vivo. Mechanistically, LEMD1-AS1 stabilized LEMD1 and increased its mRNA and protein levels, and consequently activated the PI3K-AKT signaling pathway to facilitate OSCC metastasis. Conclusions We established the lncRNA-mRNA landscape of metastatic OSCC, which indicated that LEMD1-AS1 enhanced OSCC metastasis by stabilizing its antisense transcript LEMD1. Thus, LEMD1-AS1 is a potential biomarker for predicting metastasis, as well as a therapeutic target of OSCC.

In silico identification and verification of ferroptosis-related genes in type 2 diabetic islets.

Type 2 diabetes (T2D) is a major global public health burden, with β-cell dysfunction a key component in its pathogenesis. However, the exact pathogenesis of β-cell dysfunction in T2D is yet to be fully elucidated. Ferroptosis, a recently discovered regulated form of non-apoptotic cell death, plays a vital role in the development of diabetes and its complications. The current study aimed to identify the key molecules involved in β-cell ferroptosis3 in patients with T2D using the mRNA expression profile data of GSE25724 by bioinformatic approaches. The differentially expressed mRNAs (DE-mRNAs) in human islets of patients with T2D were screened using the islet mRNA expression profiling data from the Gene Expression Omnibus and their intersection with ferroptosis genes was then obtained. Ferroptosis-related DE-mRNA functional and pathway enrichment analysis in T2D islet were performed. Using a protein-protein interaction (PPI) network constructed from the STRING database, Cytoscape software identified ferroptosis-related hub genes in the T2D islet with a Degree algorithm. We constructed a miRNA-hub gene network using the miRWalk database. We generated a rat model of T2D to assess the expression of hub genes. A total of 1,316 DE-mRNAs were identified in the islet of patients between T2D and non-T2D (NT2D), including 221 and 1,095 up- and down-regulated genes. Gene set enrichment analysis revealed that the ferroptosis-related gene set was significantly different in islets between T2D and NT2D at an overall level. A total of 33 ferroptosis-related DE-mRNAs were identified, most of which were significantly enriched in pathways including ferroptosis. The established PPI network with ferroptosis-related DE-mRNAs identified five hub genes (JUN, NFE2L2, ATG5, KRAS, and HSPA5), and the area under the ROC curve of these five hub genes was 0.929 in the Logistic regression model. We constructed a regulatory network of hub genes and miRNAs, and the results showed that suggesting that hsa-miR-6855-5p, hsa-miR-9985, and hsa-miR-584-5p could regulate most hub genes. In rat model of T2D, the protein expression levels of JUN and NFE2L2 in pancreatic tissues were upregulated and downregulated, respectively. These results contribute to further elucidation of ferroptosis-related molecular mechanisms in the pathogenesis of β-cell dysfunction of T2D.

Bioinformatics analysis and experimental verification of Notch signalling pathway-related miRNA–mRNA subnetwork in extracellular vesicles during Echinococcus granulosus encystation

BackgroundEncystation of the protoscoleces (PSCs) of Echinococcus granulosus is the main cause of secondary hydatid dissemination in the intermediate host. Extracellular vesicles (EVs) can transfer miRNAs into parasite cells to regulate mRNA expression. However, loading of developmental pathway-related miRNAs, such as those related to the Notch signalling pathway in EVs is unclear. Thus, we screened the miRNA-mRNA subnetwork involved in the Notch pathway during E. granulosus encystation in vitro and assessed changes in expression in the parasite and EVs.MethodsmRNAs and miRNAs differentially expressed (DE) between PSCs and microcysts (MCs) were screened using high-throughput sequencing. DE mRNAs obtained from transcriptome analysis were intersected with mRNAs predicted to be targets of the conserved DE miRNAs of a small RNA library. DE miRNA functions were analysed using public databases, and a miRNA–mRNA subnetwork related to the Notch pathway was established. Notch pathway-related mRNA and miRNA expression of worms and EVs at different times was verified.ResultsIn total, 1445 DE mRNAs between MCs and PSCs were screened after the intersection between 1586 DE mRNAs from the transcriptome and 9439 target mRNAs predicted using 39 DE miRNAs from the small RNA library. The DE mRNAs were clustered into 94 metabolic pathways, including the Notch pathway. Five DE miRNAs, including the most significantly expressed new DE miRNA, egr-new-mir0694-3p, corresponding to four target mRNAs (EgrG_000892700, EgrG_001029400, EgrG_001081400 and EgrG_000465800) were all enriched in the Notch pathway. The expression of the above mRNAs and miRNAs was consistent with the results of high-throughput sequencing, and the expression of each miRNA in EVs was verified. Annotated as ADAM17/TACE in the Notch pathway, EgrG_000892700 was down-regulated during PSC encystation. egr-miR-4989-3p and egr-miR-277a-3p expression in EVs after encystation was nearly five times that in EVs before encystation, which might regulate the expression of EgrG_000892700.ConclusionsFive miRNAs corresponding to four target mRNAs may be involved in regulating the Notch pathway during the PSC encystation. EVs may regulate the expression of EgrG_000892700 in PSCs because of continuous targeting of egr-miR-4989-3p and egr-miR-277a-3p and participate in the regulation the Notch pathway. The study might expand new ideas for blocking the secondary infection of E. granulosus PSCs via EVs miRNAs.Graphical

The alterations in molecular markers and signaling pathways in chronic thromboembolic pulmonary hypertension, a study with transcriptome sequencing and bioinformatic analysis.

BackgroundAt present, the alterations in molecular markers and signaling pathways in chronic thromboembolic pulmonary hypertension (CTEPH) remain unclear. We aimed to compare the difference of molecular markers and signaling pathways in patients with CTEPH and healthy people with transcriptome sequencing and bioinformatic analysis.MethodsWe prospectively included 26 patients with CTEPH and 35 sex- and age-matched healthy volunteers as control. We extracted RNA from whole blood samples to construct the library. Then, qualified libraries were sequenced using PE100 strategy on BGIseq platform. Subsequently, the DESeq2 package in R was used to screen differentially expressed mRNAs (DEmRNAs) and differentially expressed long non-coding RNAs (DElncRNAs) of 7 patients with CTEPH and 5 healthy volunteers. Afterwards, we performed functional enrichment and protein–protein interaction analysis of DEmRNAs. We also performed lncRNA-mRNA co-expression analysis and lncRNA-miRNA-mRNA network construction. In addition, we performed diagnostic analysis on the GSE130391 dataset. Finally, we performed reverse transcription polymerase chain reaction (RT-PCR) of genes in 19 patients with CTEPH and 30 healthy volunteers.ResultsGender and age between patients with CTEPH and healthy controls, between sequencing group and in vitro validation group, were comparable. A total of 437 DEmRNAs and 192 DElncRNAs were obtained. Subsequently, 205 pairs of interacting DEmRNAs and 232 pairs of lncRNA-mRNA relationship were obtained. DEmRNAs were significantly enriched in chemokine signaling pathway, metabolic pathways, arachidonic acid metabolism, and MAPK signaling pathway. Only one regulation pathway of SOBP-hsa-miR-320b-LINC00472 was found through ceRNA network construction. In diagnostic analysis, the area under curve (AUC) values of LINC00472, PIK3R6, SCN3A, and TCL6, respectively, were 0.964, 0.893, 0.750, and 0.732.ConclusionThe identification of alterations in molecules and pathways may provide further research directions on pathogenesis of CTEPH. Additionally, LINC00472, PIK3R6, SCN3A, and TCL6 may act as the potential gene markers in CTEPH.

Transcriptome analysis revealed the roles of long non-coding RNA and mRNA in the bursa of Fabricius during pigeon (Columba livia) development.

The bursa of Fabricius (BF) is the critical humoral immune organ to birds, playing an essential role in B lymphocyte differentiation. However, unlike other poultries, surgical removal of pigeon BF did not limit humoral immune responsiveness. To investigate the expression profiles and the potential role of mRNA and long non-coding RNA (LncRNA) in squab BFs, transcriptome analysis was performed by RNA-Sequencing (RNA-Seq) over three developmental stages (1-day, 13 and 26 days old). We identified 13,072 mRNAs and 19,129 lncRNAs, of which 2,752 mRNAs and 1,515 lncRNAs were differential expressed (DE) in pigeon BFs over three developmental stages. Cluster analysis presented different expression patterns in DE mRNAs and lncRNAs. Functional enrichment analysis revealed that DE lncRNAs and mRNAs with distinct expression patterns might play crucial roles in the immune system process and tissue morphogenesis. In particular, some DE genes and lncRNAs with higher expression levels in 13D or 26D are related to lymphocyte activation and differentiation, adaptive immune response, positive regulation of immune response, leukocyte migration, etc. Protein-protein interaction (PPI) network and Molecular Complex Detection (MCODE) analysis sreened six significant modules containing 37 genes from immune-related DE gene cluster, which is closely linked in B cell activation, lymphocyte differentiation, B cell receptor signaling pathway, etc. Our study characterizes mRNA and lncRNA transcriptomic variability in pigeon BFs over different developmental stages and enhances understanding of the mechanisms underlying physiological functions of pigeon BF.

The Construction and Analysis of Infiltrating Immune Cell and ceRNA Networks in Diabetic Foot Ulcer.

BackgroundDiabetic foot ulcer (DFU) is a severe complication characterized by low-grade infectious inflammation and probably associated with specific competitive endogenous RNAs (ceRNAs) and infiltrating immune cells. Nonetheless, no reliable biomarkers are used for detecting infectious inflammation in DFU. Therefore, it is essential to explore potential biomarkers for the accurate diagnosis and treatment of DFU.MethodsThe gene expression profile was retrieved from Gene Expression Omnibus (GEO) database and divided into two groups, namely, standard samples and DFU samples. To establish the ceRNA networks, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were utilized to analyze differential expression genes (DEGs). The cell type identification was achieved by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm to screen-specific immune-infiltrating cells associated with DFU.ResultsA ceRNA network was constructed with 20 differential expression circRNA (DEcircRNAs), 11 differential expression microRNAs (DEmiRNAs), and 9 differential expression mRNAs (DEmRNAs). Functional enrichment analysis demonstrated that DFU was mainly enriched in vascular endothelial growth factor (VEGF) and T-cell receptor signaling. In addition, CIBERSORT estimation indicated that CD8+ T cells and Monocytes were significantly related to the expression of IL-6, a DFU-specific infectious inflammation factor.ConclusionThis study identified that some significant ceRNAs (JUNB, GATA3, hsa-circ-0049271 and hsa-circ-0074559) and infiltrating immune cells (CD8+ T cells and monocytes) might be related to DFU infectious inflammation.

Identification of circRNA-miRNA-mRNA Regulatory Network and Crucial Signaling Pathway Axis Involved in Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is one of the most common cyanotic congenital heart diseases (CHD) worldwide; however, its pathogenesis remains unclear. Recent studies have shown that circular RNAs (circRNAs) act as “sponges” for microRNAs (miRNAs) to compete for endogenous RNA (ceRNA) and play important roles in regulating gene transcription and biological processes. However, the mechanism of ceRNA in TOF remains unclear. To explore the crucial regulatory connections and pathways of TOF, we obtained the human TOF gene, miRNA, and circRNA expression profiling datasets from the Gene Expression Omnibus (GEO) database. After data pretreatment, differentially expressed mRNAs (DEmRNAs), microRNAs (DEmiRNAs), and circRNAs (DEcircRNAs) were identified between the TOF and healthy groups, and a global triple ceRNA regulatory network, including circRNAs, miRNAs, and mRNAs based on the integrated data, was constructed. A functional enrichment analysis was performed on the Metascape website to explore the biological functions of the selected genes. Then, we constructed a protein-protein interaction (PPI) network and identified seven hub genes using the cytoHubba and MCODE plug-ins in the Cytoscape software, including BCL2L11, PIK3R1, SOCS3, OSMR, STAT3, RUNX3, and IL6R. Additionally, a circRNA–miRNA–hub gene subnetwork was established, and its enrichment analysis results indicated that the extrinsic apoptotic signaling pathway, JAK-STAT signaling pathway and PI3K-Akt signaling pathway may be involved in the pathogenesis of TOF. We further identified the hsa_circ_000601/hsa-miR-148a/BCL2L11 axis as a crucial signaling pathway axis from the subnetwork. This study provides a novel regulatory network for the pathogenesis of TOF, revealing the possible molecular mechanisms and crucial regulatory pathways that may provide new strategies for candidate diagnostic biomarkers or potential therapeutic targets for TOF.