mRNA Profiles Research Articles

Analyses of lncRNA and mRNA profiles in recurrent atrial fibrillation after catheter ablation

BackgroundAtrial fibrillation (AF) is the most common cardiac arrhythmia worldwide. Catheter ablation has become a crucial treatment for AF. However, there is a possibility of atrial fibrillation recurrence after catheter ablation. Our study sought to elucidate the role of lncRNA‒mRNA regulatory networks in late AF recurrence after catheter ablation.MethodsWe conducted RNA sequencing to profile the transcriptomes of 5 samples from the presence of recurrence after AF ablation (P-RAF) and 5 samples from the absence of recurrence after AF ablation (A-RAF). Differentially expressed genes (DEGs) and long noncoding RNAs (DE-lncRNAs) were analyzed using the DESeq2 R package. The functional correlations of the DEGs were assessed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A protein‒protein interaction (PPI) network was constructed using STRING and Cytoscape. We also established a lncRNA‒mRNA regulatory network between DE-lncRNAs and DEGs using BEDTools v2.1.2 software and the Pearson correlation coefficient method. To validate the high-throughput sequencing results of the hub genes, we conducted quantitative real-time polymerase chain reaction (qRT‒PCR) experiments.ResultsA total of 28,528 mRNAs and 42,333 lncRNAs were detected. A total of 96 DEGs and 203 DE-lncRNAs were identified between the two groups. GO analysis revealed that the DEGs were enriched in the biological processes (BPs) of “regulation of immune response” and “regulation of immune system process”, the cellular components (CCs) of “extracellular matrix” and “cell‒cell junction”, and the molecular functions (MFs) of “signaling adaptor activity” and “protein–macromolecule adaptor activity”. According to the KEGG analysis, the DEGs were associated with the “PI3K–Akt signaling pathway” and “MAPK signaling pathway.” Nine hub genes (MMP9, IGF2, FGFR1, HSPG2, GZMB, PEG10, GNLY, COL6A1, and KCNE3) were identified through the PPI network. lncRNA-TMEM51-AS1-201 was identified as a core regulator in the lncRNA‒mRNA regulatory network, suggesting its potential impact on the recurrence of AF after catheter ablation through the regulation of COL6A1, FGFR1, HSPG2, and IGF2.ConclusionsThe recurrence of atrial fibrillation after catheter ablation may be associated with immune responses and fibrosis, with the extracellular matrix playing a crucial role. TMEM51-AS1-201 has been identified as a potential key target for AF recurrence after catheter ablation.

Functional and Molecular Analysis of Human Osteoarthritic Chondrocytes Treated with Bone Marrow-Derived MSC-EVs.

Osteoarthritis (OA) is a degenerative joint disease, causing impaired mobility. There are currently no effective therapies other than palliative treatment. Mesenchymal stromal cells (MSCs) and their secreted extracellular vesicles (MSC-EVs) have shown promise in attenuating OA progression, promoting chondral regeneration, and modulating joint inflammation. However, the precise molecular mechanism of action driving their beneficial effects has not been fully elucidated. In this study, we analyzed MSC-EV-treated human OA chondrocytes (OACs) to assess viability, proliferation, migration, cytokine and catabolic protein expression, and microRNA and mRNA profiles. We observed that MSC-EV-treated OACs displayed increased metabolic activity, proliferation, and migration compared to the controls. They produced decreased proinflammatory (Il-8 and IFN-γ) and increased anti-inflammatory (IL-13) cytokines, and lower levels of MMP13 protein coupled with reduced expression of MMP13 mRNA, as well as negative microRNA regulators of chondrogenesis (miR-145-5p and miR-21-5p). In 3D models, MSC-EV-treated OACs exhibited enhanced chondrogenesis-promoting features (elevated sGAG, ACAN, and aggrecan). MSC-EV treatment also reversed the pathological impact of IL-1β on chondrogenic gene expression and extracellular matrix component (ECM) production. Finally, MSC-EV-treated OACs demonstrated the enhanced expression of genes associated with cartilage function, collagen biosynthesis, and ECM organization and exhibited a signature of 24 differentially expressed microRNAs, associated with chondrogenesis-associated pathways and ECM interactions. In conclusion, our data provide new insights on the potential mechanism of action of MSC-EVs as a treatment option for early-stage OA, including transcriptomic analysis of MSC-EV-treated OA, which may pave the way for more targeted novel therapeutics.

Spatially Preserved Multi-Region Transcriptomic Subtyping and Biomarkers of Chemoimmunotherapy Outcome in Extensive-Stage Small Cell Lung Cancer.

Transcriptomic subtyping holds promise for personalized therapy in extensive-stage small cell lung cancer (ES-SCLC). In this study, we aimed to assess intratumoral transcriptomic subtype diversity and to identify biomarkers of long-term chemoimmunotherapy benefit in human ES-SCLC. We analyzed tumor samples from 58 patients with ES-SCLC enrolled in two multicenter single-arm phase IIIb studies evaluating frontline chemoimmunotherapy in Spain: n = 32 from the IMfirst trial and n = 26 from the CANTABRICO trial. We used the GeoMx Digital Spatial Profiler system to perform multi-region transcriptomic analysis. For subtype classification, we performed hierarchical clustering using the relative expression of ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P), and YAP1 (SCLC-Y). Subtype distribution was found to be similar between bothcohorts, except for SCLC-P, which was not identified in the CANTABRICO_DSP cohort. A total of 44% of the patients in both cohorts had tumors with multiple coexisting transcriptional subtypes. Transcriptional subtypes or subtype heterogeneity was not associated with outcomes. Most potential targets did not show subtype-specific expression. Consistently in both cohorts, tumors from patients with long-term benefit (time to progression ≥12 months) contained an IFNγ-dominated mRNA profile, including enhanced capacity for antigen presentation. Hypoxia and glycolytic pathways were associated with resistance to chemoimmunotherapy. This work suggests that intratumoral heterogeneity, inconsistent association with outcome, and unclear subtype-specific target expression might be significant challenges for subtype-based precision oncology in SCLC. Preexisting IFNγ-driven immunity and mitochondrial metabolism seem to be correlates of long-term efficacy in this study, although the absence of a chemotherapy control arm precludes concluding that these are predictive features specific for immunotherapy.

Whole-Transcriptome Sequencing of Knee Joint Cartilage from Kashin-Beck Disease and Osteoarthritis Patients.

The aim of this study was to provide a comprehensive understanding of similarities and differences in mRNAs, lncRNAs, and circRNAs within cartilage for Kashin-Beck disease (KBD) compared to osteoarthritis (OA). We conducted a comparison of the expression profiles of mRNAs, lncRNAs, and circRNAs via whole-transcriptome sequencing in eight KBD and ten OA individuals. To facilitate functional annotation-enriched analysis for differentially expressed (DE) genes, DE lncRNAs, and DE circRNAs, we employed bioinformatic analysis utilizing Gene Ontology (GO) and KEGG. Additionally, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we validated the expression levels of four cartilage-related genes in chondrocytes. We identified a total of 43 DE mRNAs, 1451 DE lncRNAs, and 305 DE circRNAs in KBD cartilage tissue compared to OA (q value < 0.05; |log2FC| > 1). We also performed competing endogenous RNA network analysis, which identified a total of 65 lncRNA-mRNA interactions and 4714 miRNA-circRNA interactions. In particular, we observed that circRNA12218 had binding sites for three miRNAs targeting ACAN, while circRNA12487 had binding sites for seven miRNAs targeting COL2A1. Our results add a novel set of genes and non-coding RNAs that could potentially serve as candidate diagnostic biomarkers or therapeutic targets for KBD patients.

Exploring the immunomodulatory potential of Brahmi (Bacopa monnieri) in the treatment of invasive ductal carcinoma.

Bacopa monnieri (L) Wettst, commonly known as Brahmi, stands as a medicinal plant integral to India's traditional medical system, Ayurveda, where it is recognized as a "medhya rasayana"-a botanical entity believed to enhance intellect and mental clarity. Its significant role in numerous Ayurvedic formulations designed to address conditions such as anxiety, memory loss, impaired cognition, and diminished concentration underscores its prominence. Beyond its application in cognitive health, Brahmi has historically been employed in Ayurvedic practices for the treatment of inflammatory diseases, including arthritis. In contemporary biomedical research, Bacopa monnieri can attenuate the release of pro-inflammatory cytokines TNF-α and IL-6 in animal models. However, there remains a paucity of information regarding Bacopa's potential as an anticancer agent, warranting further investigation in this domain. Based on previous findings with Brahmi (Bacopa monnieri), the current study aims to find out the role of Brahmi plant preparation (BPP) in immunomodulatory actions on IDC. Employing a specific BPP concentration, we conducted a comprehensive study using MTT assay, ELISA, DNA methylation analysis, Western blotting, ChIP, and mRNA profiling to assess BPP's immunomodulatory properties. Our research finding showed the role of BPP in augmenting the action of T helper 1 (TH1) cells which secreted interferon-γ (IFN-γ) which in turn activated cytotoxic T-lymphocytes (CTL) to kill the cells of IDC (*p < 0.05). Moreover, we found out that treatment with BPP not only increased the activities of tumor-suppressor genes (p53 and BRCA1) but also decreased the activities of oncogenes (Notch1 and DNAPKcs) in IDC (*p < 0.05). BPP had an immense significance in controlling the epigenetic dysregulation in IDC through the downregulation of Histone demethylation & Histone deacetylation and upregulation of Histone methylation and Histone acetylation (*p < 0.05). Our Chromatin immunoprecipitation (ChIP)-qPCR data showed BPP treatment increased percentage enrichment of STAT1 & BRCA1 (*p < 0.05) and decreased percentage enrichment of STAT3, STAT5 & NF ΚB (*p < 0.05) on both TBX21 and BRCA1 gene loci in IDC. In addition, BPP treatment reduced the hypermethylation of the BRCA1-associated-DNA, which is believed to be a major factor in IDC (*p < 0.05). BPP not only escalates the secretion of type 1 specific cytokines but also escalates tumor suppression and harmonizes various epigenetic regulators and transcription factors associated with Signal Transducer and Activator of Transcription (STAT) to evoke tumor protective immunity in IDC.

Evaluation of EGFR and COX pathway inhibition in human colon organoids of serrated polyposis and other hereditary cancer syndromes.

Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs.

Construction and analysis of competitive endogenous RNA networks and prognostic models associated with ovarian cancer based on the exoRBase database.

To construct a competitive endogenous RNA (ceRNA) regulatory network in blood exosomes of patients with ovarian cancer (OC) using bioinformatics and explore its pathogenesis. The exoRbase2.0 database was used to download blood exosome gene sequencing data from patients OC and normal controls and the expression profiles of exosomal mRNA, long non-coding RNA (lncRNA), and circular RNA (circRNA) were detected independently using R language for differential expression analysis. TargetScan and miRanda databases were combined for the prediction and differential expression of mRNA-binding microRNAs (miRNA). The miRcode and starBase databases were used to predict miRNAs that bind to differentially expressed lncRNAs and circRNAs repectively. The relevant mRNA, circRNA, lncRNA and their corresponding miRNA prediction data were imported into Cytoscape software for visualization of the ceRNA network. The R language and KEGG Orthology-based Annotation System (KOBAS) were used to execute and illustrate the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Hub genes were identified using The CytoHubba plugin. Thirty-one differentially expressed mRNAs, 17 differentially expressed lncRNAs, and 24 differentially expressed circRNAs were screened. Cytoscape software was used to construct the ceRNA network with nine mRNA nodes, two lncRNA nodes, eight circRNA nodes, and 51 miRNA nodes. Both GO and KEGG were focused on the Spliceosome pathway, indicating that spliceosomes are closely linked with the development of OC, while heterogenous nuclear ribonucleoprotein K and RNA binding motif protein X-linked genes were the top 10 score Hub genes screened by Cytoscape software, including two lncRNAs, four mRNAs, and four circRNAs. In patients with OC, the expression of eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), SERPINE 1 mRNA binding protein 1 (SERBP1), ribosomal protein L15 (RPL15) and human leukocyte antigen complex P5 (HCP5) was significantly higher whereas that of testis expressed transcript, Y-linked 15 and DEAD-box helicase 3 Y-linked genes was lower compared to normal controls Immunocorrelation scores revealed that SERBP1 was significantly and negatively correlated with endothelial cells and CD4+ T cells and positively correlated with natural killer (NK) cells and macrophages, respectively; RPL15 was significantly positively correlated with macrophages and endothelial cells and negatively correlated with CD8+ T cells and uncharacterized cells, respectively. EIF4G2 was significantly and negatively correlated with endothelial cells and CD4+ T cells, and positively correlated with uncharacterized cells, respectively. Based on the survival data and the significant correlation characteristics derived from the multifactorial Cox analysis (P < 0.05), the survival prediction curves demonstrated that the prognostic factors associated with 3-year survival in patients with OC were The prognostic factors associated with survival were Macrophage, RPL15. This study successfully constructs a ceRNA regulatory network in blood exosomes of OV patients, which provides the specific targets for diagnosis and treatment of OC.

Long noncoding RNAs and mRNAs profiling in ovary during laying and broodiness in Taihe Black-Bone Silky Fowls (Gallus gallus Domesticus Brisson).

Broodiness significantly impacts poultry egg production, particularly notable in specific breeds such as the black-boned Silky, characterized by pronounced broodiness. An understanding of the alterations in ovarian signaling is essential for elucidating the mechanisms that influence broodiness. However, comparative research on the characteristics of long non-coding RNAs (lncRNAs) in the ovaries of broody chickens (BC) and high egg-laying chickens (GC) remains scant. In this investigation, we employed RNA sequencing to assess the ovarian transcriptomes, which include both lncRNAs and mRNAs, in eight Taihe Black-Bone Silky Fowls (TBsf), categorized into broody and high egg-laying groups. This study aims to provide a clearer understanding of the genetic underpinnings associated with broodiness and egg production. We have identified a total of 16,444 mRNAs and 18,756 lncRNAs, of which 349 mRNAs and 651 lncRNAs exhibited significantly different expression (DE) between the BC and GC groups. Furthermore, we have identified the cis-regulated and trans-regulated target genes of differentially abundant lncRNA transcripts and have constructed an lncRNA-mRNA trans-regulated interaction network linked to ovarian follicle development. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analyses have revealed that DE mRNAs and the target genes of DE lncRNAs are associated with pathways including neuroactive ligand-receptor interaction, CCR6 chemokine receptor binding, G-protein coupled receptor binding, cytokine-cytokine receptor interaction, and ECM-receptor interaction. Our research presents a comprehensive compilation of lncRNAs and mRNAs linked to ovarian development. Additionally, it establishes a predictive interaction network involving differentially abundant lncRNAs and differentially expressed genes (DEGs) within TBsf. This significantly contributes to our understanding of the intricate interactions between lncRNAs and genes governing brooding behavior.

Identification of a molecular network regulated by multiple ASD high risk genes.

Genetic sequencing has identified high-confidence ASD risk genes with loss-of-function mutations. How the haploinsufficiency of distinct ASD risk genes causes ASD remains to be elucidated. In this study, we examined the role of four top-ranking ASD risk genes, ADNP, KDM6B, CHD2, and MED13, in gene expression regulation. ChIP-seq analysis reveals that gene targets with the binding of these ASD risk genes at promoters are enriched in RNA processing and DNA repair. Many of these targets are found in ASD gene database (SFARI), and are involved in transcription regulation and chromatin remodeling. Common gene targets of these ASD risk genes include a network of high confidence ASD genes associated with gene expression regulation, such as CTNNB1 and SMARCA4. We further directly examined the transcriptional impact of the deficiency of these ASD risk genes. Our mRNA profiling with qPCR assays in cells with the knockdown of Adnp, Kdm6b, Chd2 or Med13 has revealed an intricate pattern of their cross-regulation, as well as their influence on the expression of other ASD genes. In addition, some synaptic genes, such as Snap25 and Nrxn1, are strongly regulated by deficiency of the four ASD risk genes, which could be through the direct binding at promoters or indirectly through the targets like Ctnnb1 or Smarca4. The identification of convergent and divergent gene targets that are regulated by multiple ASD risk genes will help to understand the molecular mechanisms underlying common and unique phenotypes associated with haploinsufficiency of ASD-associated genes.

Whole transcriptome sequencing reveals key genes and ceRNA regulatory networks associated with pimpled eggs in hens

Eggshell is one of the most important indicators of egg quality, and due to low shell strength, pimple eggs (PE) are more susceptible to breakage, thus causing huge economic losses to the egg industry. At the current time, the molecular mechanisms that regulate the formation of pimple eggs are poorly understood. In this study, uterine tissues of PE-laying hens (n = 8) and normal egg (NE) -laying hens (n = 8) were analyzed by whole transcriptome sequencing, and a total of 619 differentially expressed mRNAs (DE mRNAs), 122 differentially expressed lncRNAs (DE lncRNAs) and 21 differentially expressed miRNAs (DE miRNAs) were obtained. Based on the targeting relationship among DE mRNAs, DE lncRNAs and DE miRNAs, we constructed a competitive endogenous RNA (ceRNA) network including 12 DEmiRNAs, 19 DElncRNAs and 128 DEmRNAs. Considering the large amount of information contained in the network, we constructed a smaller ceRNA network to better understand the complex mechanisms of pimple egg formation. The smaller ceRNA network network contains 7 DE lncRNAs (LOC107056551, LOC121109367, LOC121108909, LOC121108862, LOC112530033, LOC121113165, LOC107054145), 5 DE miRNAs (gga-miR-6568-3p, gga-miR-31-5p, gga-miR-18b-3p, gga-miR-1759-3p, gga-miR-12240-3p) and 7 DE mRNAs (CABP1, DNAJC5, HCN3, HPCA, IBSP, KCNT1, OTOP3), and these differentially expressed genes may play key regulatory roles in the formation of pimpled eggs in hens. This study provides the overall expression profiles of mRNAs, lncRNAs and miRNAs in the uterine tissues of hens, which provides a theoretical basis for further research on the molecular mechanisms of pimpled egg formation, and has potential applications in improving eggshell quality.

Comprehensive analysis of differentially expressed mRNA profiles in chicken jejunum and cecum following Eimeria maxima infection

Coccidiosis, a protozoan disease that substantially impacts poultry production, is characterized by an intracellular parasite. The study utilized 48 one-day-old Horro chickens, randomly divided into the infected (I) and control (C) groups. The challenge group of chickens were administered Eimeria maxima oocysts via oral gavage at 21 days old, and each chicken received 2ml containing 7×104 sporulated oocysts. The total RNAs of chicken jejunum and cecum tissues were isolated from three samples, each from I and C groups. Our study aimed to understand the host immune-parasite interactions and compare immune response mRNA profiles in chicken jejunum and cecum tissues at 4 and 7 days post-infection with Eimeria maxima. The results showed that 823 up- and 737 down-regulated differentially expressed mRNA (DEmRNA) in jejunum at 4 days infection and control (J4I verses J4C), and 710 up- and 368 down-regulated DEGs in jejunum at 7 days infection and control (J7I verses J7C) were identified. In addition, DEmRNA in cecum tissue, 1424 up- and 1930 down-regulated genes in cecum at 4 days infection and control (C4I verses C4C), and 77 up- and 191 down-regulated genes in cecum at 7 days infection and control (C7I verses C7C) were detected. The crucial DEmRNAs, including SLC7A5, IL1R2, GLDC, ITGB6, ADAMTS4, IL1RAP, TNFRSF11B, IMPG2, WNT9A, and FOXF1, played pivotal roles in the immune response during Eimeria maxima infection of chicken jejunum. In addition, the potential detection of FSTL3, RBP7, CCL20, DPP4, PRKG2, TFPI2, and CDKN1A in the cecum during the host immune response against Eimeria maxima infection is particularly noteworthy. Furthermore, our functional enrichment analysis revealed the primary involvement of DEmRNAs in small molecule metabolic process, immune response function, inflammatory response, and toll-like receptor 10 signaling pathway in the jejunum at 4 and 7 days post-infection. Similarly, in the cecum, DEmRNAs at 4 and 7 days post-infection were enriched in processes related to oxidative stress response and immune responses. Our findings provide new insights and contribute significantly to the field of poultry production and parasitology.

Identification of N7-methylguanosine-related miRNAs as potential biomarkers for prognosis and drug response in breast cancer

ObjectivesThe impact of N7-methylguanosine (m7G) on tumor progression and the regulatory role of microRNAs (miRNAs) in immune function significantly influence breast cancer (BC) prognosis. Investigating the interplay between m7G modification and miRNAs provides novel insights for assessing prognostics and drug responses in BC. Materials and methodsRNA sequences (miRNA and mRNA profiles) and clinical data for BC were acquired from the Cancer Genome Atlas (TCGA) database. A miRNA signature associated with 15 m7G in this cohort was identified using Cox regression and LASSO. The risk score model was evaluated using Kaplan-Meier and time-dependent ROC analysis, categorizing patients into high-risk and low-risk groups. Functional enrichment analyses were conducted to explore potential pathways. The immune system, including scores, cell infiltration, function, and drug sensitivity, was examined and compared between high-risk and low-risk groups. A nomogram that combines risk scores and clinical factors was developed and validated. Single-sample gene set enrichment analysis (ssGSEA) was employed to explore m7G-related miRNA signatures and immune cell relationships in the tumor microenvironment. Additionally, drug susceptibility was compared between risk groups. ResultsFifteen m7G-related miRNAs were independently correlated with overall survival (OS) in BC patients. Time-dependent ROC analysis yielded area under the curve (AUC) values of 0.742, 0.726, and 0.712 for predicting 3-, 5-, and 10-year survival rates, respectively. The Kaplan-Meier analysis revealed a significant disparity in OS between the high-risk and low-risk groups (p = 1.3e-6). Multiple regression identified the risk score as a significant independent prognostic factor. An excellent calibration nomogram with a C-index of 0.785 (95 % CI: 0.728–0.843) was constructed. In immune analysis, low-risk patients exhibited heightened immune function and increased responsiveness to immunotherapy and chemotherapy compared to high-risk patients. ConclusionThis study systematically analyzed m7G-related miRNAs and revealed their regulatory mechanisms concerning the tumor microenvironment (TME), pathology, and the prognosis of BC patient. Based on these miRNAs, a prognostic model and nomogram were developed for BC patients, facilitating prognostic assessments. These findings can also assist in predicting treatment responses and guiding medication selection.

474 An Investigation of Novel Urinary Cell mRNA Profiles for Noninvasive Diagnosis of Acute Rejection in Kidney Transplant Recipients

OBJECTIVES/GOALS: RNA-seq of urine and kidney allograft biopsies (bx) found that urinary cell immune landscape reflects intragraft molecular events and we discovered a shared set of 127 mRNAs in urine matched to T cell mediated and antibody mediated rejection bx. We prioritized ITM2A, SLAMF6 and IKZF3 mRNAs and herein investigate if these accurately predict rejection. METHODS/STUDY POPULATION: We collected urine samples from adult kidney allograft (KA) recipients at the time of KA bx. KA bx were classified by pathologists by Banff criteria. Total RNA was isolated from KA bx-matched urine samples. Absolute copy numbers of ITM2A, SLAMF6, and IKZF3 mRNAs and 18S rRNA were measured using our customized RT-qPCR assays. Logistic regression used to derive an equation for a combined signature score of 18S-normalized urinary cell mRNA levels of ITM2A, IKZF3, and SLAMF6 that best predicts Acute Rejection (AR= both T cell mediated rejection and antibody mediated rejection). Area under the ROC curve (AUC) was calculated to discriminate between AR and No Rejection (NR) biopsies for 18S-normalized urinary cell levels of ITM2A, IKZF3 and SLAMF6 and the composite signature score. AUCs were compared by DeLong Method. RESULTS/ANTICIPATED RESULTS: Urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNAs in urine discriminated KA recipients with AR biopsies (n=95) from those with NR biopsies (n=160) (All P values &lt;0.05, Mann-Whitney test) and the AUC was 0.69 (95%CI, 0.62 to 0.76) for ITM2A, 0.61 (95%CI, 0.53 to 0.68) for IKZF3, and 0.60 (95%CI, 0.53 to 0.68) for SLAMF6. The derived combination signature score of urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNA discriminated KA recipients with AR from those with NR (P&lt;0.0001, Mann Whitney test) and the combined signature score AUC was 0.72 (95%CI, 0.65 to 0.79). The combination signature score discriminated AR vs NR better than IKZF3 and SLAMF6 alone, but was not significantly different than ITM2A alone (DeLong method). (Additional results/figures to be included in poster) DISCUSSION/SIGNIFICANCE: Our RNA-seq offered a unique opportunity to diagnose AR by measuring the mRNAs in urine. We now found that urinary cell mRNA levels of ITM2A, IKZF3, SLAMF6 and the combined signature are diagnostic of AR, a major and serious post-transplant complication. This allows for much-needed KA molecular surveillance and personalization of immunosuppression.

Expression profile of mRNAs and miRNAs related to mitogen-activated kinases in HaCaT cell culture treated with lipopolysaccharide a and adalimumab

ABSTRACT Studies indicate that mitogen-activated protein kinases (MAPKs) exhibit activation and overexpression within psoriatic lesions. This study aimed to investigate alterations in the expression patterns of genes encoding MAPKs and microRNA (miRNA) molecules that potentially regulate their expression in human adult low-calcium high-temperature (HaCaT) keratinocytes when exposed to bacterial lipopolysaccharide A (LPS) and adalimumab. HaCaT cells underwent treatment with 1 µg/mL LPS for 8 hours, followed by treatment with 8 µg/mL adalimumab for 2, 8, or 24 hours. Untreated cells served as controls. The molecular analysis involved microarray, quantitative real-time polymerase chain reaction (RTqPCR), and enzyme-linked immunosorbent assay (ELISA) analyses. Changes in the expression profile of seven mRNAs: dual specificity phosphatase 1 (DUSP1), dual specificity phosphatase 3 (DUSP3), dual specificity phosphatase 4 (DUSP4), mitogen-activated protein kinase 9 (MAPK9), mitogen-activated protein kinase kinase kinase 2 (MAP3K2), mitogen-activated protein kinase kinase 2 (MAP2K2), and MAP kinase-activated protein kinase 2 (MAPKAPK2, also known as MK2) in cell culture exposed to LPS or LPS and the drug compared to the control. It was noted that miR-34a may potentially regulate the activity of DUSP1, DUSP3, and DUSP4, while miR-1275 is implicated in regulating MAPK9 expression. Additionally, miR-382 and miR-3188 are potential regulators of DUSP4 levels, and miR-200-5p is involved in regulating MAPKAPK2 and MAP3K2 levels. Thus, the analysis showed that these mRNA molecules and the proteins and miRNAs they encode appear to be useful molecular markers for monitoring the efficacy of adalimumab therapy.

Whole transcriptome mapping reveals the lncRNA regulatory network of TFP5 treatment in diabetic nephropathy.

TFP5 is a Cdk5 inhibitor peptide, which could restore insulin production. However, the role of TFP5 in diabetic nephropathy (DN) is still unclear. This study aims to characterize the transcriptome profiles of mRNA and lncRNA in TFP5-treated DN mice to mine key lncRNAs associated with TFP5 efficacy. We evaluated the role of TFP5 in DN pathology and performed RNA sequencing in C57BL/6Jcontrol mice, C57BL/6J db/db model mice, and TFP5 treatment C57BL/6J db/db model mice. The differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) were analyzed. WGCNA was used to screen hub-gene of TFP5 in treatment of DN. Our results showed that TFP5 therapy ameliorated renal tubular injury in DN mice. In addition, compared with the control group, the expression profile of lncRNAs in the model group was significantly disordered, while TFP5 alleviated the abnormal expression of lncRNAs. A total of 67 DElncRNAs shared among the three groups, 39 DElncRNAs showed a trend of increasing in the DN group and decreasing after TFP treatment, while the remaining 28 showed the opposite trend. DElncRNAs were enriched in glycosphingolipid biosynthesis signaling pathways, NF-κB signaling pathways, and complement activation signaling pathways. There were 1028 up-regulated and 1117 down-regulated DEmRNAs inthe model group compared to control group, and 123 up-regulated and 153 down-regulated DEmRNAs in theTFP5 group compared to themodel group. The DEmRNAs were involved in PPAR and MAPK signaling pathway. We confirmed that MSTRG.28304.1 is a key DElncRNA for TFP5 treatment of DN. TFP5 ameliorated DN maybe by inhibiting MSTRG.28304.1 through regulating the insulin resistance and PPAR signaling pathway. The qRT-PCR results confirmed the reliability of the sequencing data through verifying the expression of ENSMUST00000211209, MSTRG.31814.5, MSTRG.28304.1, and MSTRG.45642.14. Overall, the present study provides novel insights into molecular mechanisms of TFP5 treatment in DN.

LncRNA and mRNA expression characteristic and bioinformatic analysis in myocardium of diabetic cardiomyopathy mice

BackgroundDiabetic cardiomyopathy (DCM) is becoming a very well-known clinical entity and leads to increased heart failure in diabetic patients. Long non-coding RNAs (LncRNAs) play an important role in the pathogenesis of DCM. In the present study, the expression profiles of lncRNAs and mRNAs were illuminated in myocardium from DCM mice, with purpose of exploring probable pathological processes of DCM involved by differentially expressed genes in order to provide a new direction for the future researches of DCM.ResultsThe results showed that a total of 93 differentially expressed lncRNA transcripts and 881 mRNA transcripts were aberrantly expressed in db/db mice compared with the controls. The top 6 differentially expressed lncRNAs like up-regulated Hmga1b, Gm8909, Gm50252 and down-regulated Msantd4, 4933413J09Rik, Gm41414 have not yet been reported in DCM. The lncRNAs-mRNAs co-expression network analysis showed that LncRNA 2610507I01Rik, 2310015A16Rik, Gm10503, A930015D03Rik and Gm48483 were the most relevant to differentially expressed mRNAs.ConclusionOur results showed that db/db DCM mice exist differentially expressed lncRNAs and mRNAs in hearts. These differentially expressed lncRNAs may be involved in the pathological process of cardiomyocyte apoptosis and fibrosis in DCM.

Construction of the underlying circRNA-miRNA-mRNA regulatory network and a new diagnostic model in ulcerative colitis by bioinformatics analysis.

Circular RNAs (circRNAs) are involved in the pathogenesis of many diseases through competing endogenous RNA (ceRNA) regulatory mechanisms. To investigate a circRNA-related ceRNA regulatory network and a new predictive model by circRNA to understand the diagnostic mechanism of circRNAs in ulcerative colitis (UC). We obtained gene expression profiles of circRNAs, miRNAs, and mRNAs in UC from the Gene Expression Omnibus dataset. The circRNA-miRNA-mRNA network was constructed based on circRNA-miRNA and miRNA-mRNA interactions. Functional enrichment analysis was performed to identify the biological mechanisms involved in circRNAs. We identified the most relevant differential circRNAs for diagnosing UC and constructed a new predictive nomogram, whose efficacy was tested with the C-index, receiver operating characteristic curve (ROC), and decision curve analysis (DCA). A circRNA-miRNA-mRNA regulatory network was obtained, containing 12 circRNAs, three miRNAs, and 38 mRNAs. Two optimal prognostic-related differentially expressed circRNAs, hsa_circ_0085323 and hsa_circ_0036906, were included to construct a predictive nomogram. The model showed good discrimination, with a C-index of 1(> 0.9, high accuracy). ROC and DCA suggested that the nomogram had a beneficial diagnostic ability. This novel predictive nomogram incorporating hsa_circ_0085323 and hsa_circ_0036906 can be conveniently used to predict the risk of UC. The circRNa-miRNA-mRNA network in UC could be more clinically significant.

Analysis of differentially expressed lncRNAs and mRNAs associated with slow‑transit constipation

Slow transit constipation (STC) is a refractory gastrointestinal disease, accounting for approximately 13 ∼ 37 % of chronic constipation. However, the molecular mechanism of STC remains poorly understood. Herein, this study aims to identify the key mRNAs and lncRNAs associated with STC. To this end, we performed high-throughput RNA sequencing to identify differentially expressed (DE) mRNAs and lncRNAs in the whole-layer sigmoid intestinal tissues from 4 STC patients and 4 non-STC patients. The identified DE lncRNAs and mRNAs were validated through quantitative real-time PCR. Weighted gene co-expression network analysis (WGCNA) and Pearson correlation analysis were conducted to determine the significantly correlated DE mRNA-lncRNA pairs. A total of 1420 DE lncRNAs and 1634 DE mRNAs were identified. Kyoto Encyclopedia of Genes and Genomes analysis of DE mRNAs indicated that these DE mRNAs might be associated with systemic lupus erythematosus, alcoholism, intestinal immune network for IgA production, inflammatory bowel disease, NF-kappa B signaling pathway. WGCNA and Pearson correlation analyses jointly identified 16,577 significantly correlated DE mRNA-lncRNA pairs. Furthermore, lncRNAs LINC00641, LINC02268, LINC03013 were identified as hub lncRNAs. The protein–protein interaction (PPI) network of proteins encoded by DE mRNAs was established, and PPI-based analysis revealed that Interleukin 2(IL2), CD80 molecule (CD80), interleukin-17A (IL-17A) might play significant roles in the development of STC. This study analyzes the expression profiles of lncRNAs and mRNAs associated with STC. Our findings will contribute to further understanding of the molecular mechanism of STC and provide potential diagnostic or therapeutic biomarkers for STC.

Identification of Plasma Exosomes hsa_circ_0001360 and hsa_circ_0000038 as Key Biomarkers of Coronary Heart Disease.

Coronary heart disease (CHD) is the leading cause of death and disability worldwide. Accumulating evidence reveals that atherosclerosis (AS), characterized by systemic, chronic, and multifocal disease, and is the primary pathological basis of cardiovascular diseases, including CHD. However, the molecular underpinnings of CHD are still far from well understood. Our study attempted to identify aberrant plasma exosome-derived circRNAs and key exosomal circRNA biomarkers for CHD. The expression profiles of mRNAs, circRNAs, and lncRNAs in the blood exosomes of CHD patients and healthy controls were obtained from the exoRBase database. The corresponding miRNAs of the differentially expressed mRNAs, circRNAs, and lncRNAs were predicted via ENCORI and the miRcode database. LncRNAs/circRNAs and mRNAs with the cotargeted miRNAs were selected to construct an interaction network. Multiple machine learning algorithms have been used to explore potential biomarkers, followed by verification in patients with CHD using real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Based on the cutoff criterion of P < 0.05, we identified 85 differentially expressed circRNAs (4 upregulated and 81 downregulated), 43 differentially expressed lncRNAs (24 upregulated and 19 downregulated), and 312 differentially expressed mRNAs (55 upregulated and 257 downregulated). Functional enrichment analysis revealed that the differentially expressed mRNAs were involved mainly in neutrophil extracellular trap (NET) formation and the nucleotide-binding oligomerization domain- (NOD-) like receptor signaling pathway. Further analysis revealed that the DEGs in the circRNA/lncRNA-miRNA-mRNA interaction network were closely related to lipid and atherosclerotic signaling pathways. Hsa_circ_0001360 and hsa_circ_0000038 were identified as potential biomarkers for CHD based on three machine learning algorithms. The relative expression levels of hsa_circ_0001360 and hsa_circ_0000038 were significantly altered in plasma exosomes from patients with CHD. ROC curve analysis revealed that the areas under the curve (AUCs) were 0.860, 0.870, and 0.940 for hsa_circ_0001360, hsa_circ_0000038, and the two-gene combination, respectively. The circRNA/lncRNA-miRNA-mRNA interaction network might help to elucidate the pathogenesis of CHD. Hsa_circ_0001360 combined with hsa_circ_0000038 might be an important diagnostic biomarker.

Abstract 5249: Macrophages expressing synthetic cytokine receptors reverse IL10-mediated immunosuppression within solid tumors and promote adaptive immunity

Abstract Motivation: In solid tumors, anti-inflammatory cytokines such as IL10 and TGFβ support an immunosuppressive tumor microenvironment (TME) and inhibit anti-tumor immunity. Many therapeutic approaches to overcome immunosuppression have used monoclonal antibodies to block cytokine signaling. Instead of silencing TME-associated cytokines, a more powerful strategy may be to leverage them as disease markers and convert them into anti-tumor signals, using a logic-gated cell-based immunotherapy. Here, we engineered macrophages with synthetic cytokine switch receptors (SR) that convert IL10 or TGFβ into pro-inflammatory signals. Macrophages are homeostatic regulators capable of both infiltrating solid tumors and initiating inflammation, and we harnessed this proficiency using SRs that convert prevalent cytokines in the TME into pro-inflammatory responses for TME modulation. We termed this engineered myeloid cell platform “Engineered Microenvironment Converters” (EM-C) and evaluated its ability to overcome cytokine-mediated immunosuppression in solid tumors. Methods: EM-C were produced by transducing primary macrophages or monocytes with a SR that converts IL10 into an interferon-based signal. The response of EM-C to IL10 was monitored in vitro using phenotypic characterization of surface molecules, measurement of secreted factors, and mRNA profiling. The in vivo activity of EM-C was evaluated using a subcutaneous tumor model in immunocompetent mice. Murine EM-C were administered to tumor-bearing mice, and their ability to modulate the TME was monitored via immunophenotyping, secretome analysis, and single cell transcriptomics. To demonstrate modularity of the EM-C platform, additional SR were designed to convert TGFβ into interferon or toll-like receptor signals. Results: EM-C efficiently sequestered IL10 or TGFβ and upregulated pro-inflammatory markers, cytokines, and pathways in a dose-dependent manner in vitro. EM-C administered to tumor-bearing mice remodeled the TME immune compartment by increasing the abundance of CD8 T cells and reducing the presence of immunosuppressive Tregs. EM-C furthermore augmented the cytokine/chemokine profile of the TME in a manner that correlated with anti-tumor response. TME modulation by EM-C improved tumor control, as compared to non-engineered macrophages, and had additive efficacy with checkpoint blockade. Conclusion: We present a versatile immunotherapy platform that harnesses macrophages as “living converters” to locally augment inflammation in solid tumors. EM-C exhibit a modular ability to locally convert IL10 or TGFβ into pro-inflammatory signals without systemic cytokine antagonism. The EM-C platform enables development of target antigen-agnostic myeloid cell immunotherapies for overcoming immunosuppression in diverse solid tumors. Citation Format: Chris Sloas, Silvia Beghi, Yuhao Huangfu, Rehman Qureshi, Benjamin Schott, Michael Ball, Daniel Blumenthal, Thomas Condamine, Michael Klichinsky, Yumi Ohtani. Macrophages expressing synthetic cytokine receptors reverse IL10-mediated immunosuppression within solid tumors and promote adaptive immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5249.