RNA Profiling Research Articles

FLASH Proton Radiation Therapy Mitigates Inflammatory and Fibrotic Pathways and Preserves Cardiac Function in a Preclinical Mouse Model of Radiation-Induced Heart Disease

PurposeStudies during the past 9 years suggest that delivering radiation at dose rates exceeding 40 Gy/s, known as “FLASH” radiotherapy, enhances the therapeutic index of radiation therapy (RT) by decreasing normal tissue damage while maintaining tumor response compared to conventional (or standard) RT. This study demonstrates the cardioprotective benefits of FLASH proton RT (F-PRT) compared to standard (conventional) proton RT (S-PRT), as evidenced by reduced acute and chronic cardiac toxicities. Methods and MaterialsMice were imaged using cone beam computed tomography to precisely determine the heart's apex as the beam isocenter. Irradiation was conducted utilizing a shoot-through technique with a 5mm diameter circular collimator. Bulk RNA-sequencing was performed on non-irradiated (NR) samples, as well as apexes treated with F-PRT or S-PRT, at 2 weeks following a single 40 Gy dose. Inflammatory responses were assessed through multiplex cytokine/chemokine microbead assay and immunofluorescence analyses. Levels of perivascular fibrosis were quantified using Masson's Trichrome and Picrosirius red staining. Additionally, cardiac tissue functionality was evaluated by 2D echocardiograms at 8 and 30 weeks post-PRT. ResultsRadiation damage was specifically localized to the heart's apex. RNA profiling of cardiac tissues treated with PRT revealed that S-PRT uniquely upregulated pathways associated with DNA damage response, induction of tumor necrosis factor superfamily and inflammatory response, while F-PRT primarily affected cytoplasmic translation, mitochondrion organization and ATP synthesis. Notably, F-PRT led to a milder inflammatory response, accompanied by significantly attenuated changes in TGF-β1 and αSMA levels. Critically, F-PRT decreased collagen deposition, and better preserved cardiac functionality compared to S-PRT. ConclusionsThis study demonstrated that F-PRT reduces the induction of an inflammatory environment with lower expression of inflammatory cytokines and profibrotic factors. Importantly, the results indicate that F-PRT better preserves cardiac functionality, as confirmed by echocardiography analysis, while also mitigating the development of long-term fibrosis.

Effects of non-nutritive sweeteners on growth and intestinal health by regulating hypothalamic RNA profile and ileum microbiota in guinea pigs.

Non-nutritive sweeteners (NNS) are commonly used in sweetened foods and beverages; however their role in metabolic regulation is still not clear. In this experiment, we used guinea pigs as an animal model to study the effect of NNS on body growth and intestinal health by modifying gut microbiota and hypothalamus-related proteins. For a 28-day feeding experiment a total of 40 guinea pigs were randomly divided into four groups, one control (CN) group and three treatments, in which three NNS were added to the diet: rebaudioside A (RA, 330 mg kg-1), sodium saccharin (SS, 800 mg kg-1), and sucralose (TGS, 167 mg kg-1), respectively. The TGS group exhibited significantly reduced food consumption in comparison with the CN group (P < 0.05) whereas the RA group showed increased food consumption in comparison with the CN group (P < 0.05). Notably, Taste receptor type 1 subunit 2 (T1R2) expression in the hypothalamus was significantly higher in the RA group than in the CN group (P < 0.05). The mRNA expressions of appetite-stimulated genes arouti-related neuropeptide (AGRP), neuropeptide Y (NPY), and thyroid stimulating hormone (TSHB) were significantly higher than those in the CN group (P < 0.05) but mRNA expressions of appetite-suppressed genes tryptophan hydroxylase 2(THP2) were significantly lower in the TGS group (P < 0.05). Furthermore, NNS in the guinea pig diets (RA, SS, TGS) significantly increased the relative abundance of Muribaculaceae but decreased the relative abundance of Clostridia_vadin BB60 in comparison with the CN group (P < 0.05). We also found that dietary supplementation with RA also significantly altered the relative abundance of Lactobacillus. Our finding confirmed that dietary supplementation with RA and TGS affected body growth and intestinal health by modulating hypothalamic RNA profiles and ileum microbiota, suggesting that NNS should be included in guinea-pig feeding. © 2024 Society of Chemical Industry.

Genome-wide transcriptional profiling and functional analysis of long noncoding RNAs and mRNAs in chicken macrophages associated with the infection of avian pathogenic E. coli

BackgroundAvian pathogenic E. coli (APEC) can cause localized or systemic infections, collectively known as avian colibacillosis, resulting in huge economic losses to poultry industry globally per year. In addition, increasing evidence indicates that long non-coding RNAs (lncRNAs) play a critical role in regulating host inflammation in response to bacterial infection. However, the role of lncRNAs in the host response to APEC infection remains unclear.ResultsHere, we found 816 differentially expressed (DE) lncRNAs and 1,798 DE mRNAs in APEC infected chicken macrophages by RNAseq. The identified DE lncRNA-mRNAs were involved in Toll like receptor signaling pathway, VEGF signaling pathway, fatty acid metabolism, phosphatidylinositol signaling system, and other types of O-glycan biosynthesis. Furthermore, we found the novel lncRNA TCONS_00007391 as an important immune regulator in APEC infection was able to regulate the inflammatory response by directly targeting CD86.ConclusionThese findings provided a better understanding of host response to APEC infection and also offered the potential drug targets for therapy development against APEC infection.

Circulating serum profile of small non-coding RNAs in patients with anaphylaxis beyond microRNAs.

Anaphylaxis is the most severe manifestation of allergic disorders. Currently, an increasing number of cells, pathways and molecules involved in the etiopathogenesis of anaphylaxis are being discovered. However, there are no conclusive biomarkers to confirm its diagnosis. Small non-coding RNAs (sncRNAs) are 18-200 nucleotide molecules that can be divided into: microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), transference RNA derived fragments (tRFs) and YRNA derived fragments (YRFs). These molecules participate in cell-cell communication modulating various physiological processes and have been postulated as non-invasive biomarkers of several pathologies. Therefore, in this study we characterized the serum circulating profile of other sncRNA beyond miRNAs in two populations of 5 adults and 5 children with drug- and food-mediated anaphylaxis, respectively. Samples were obtained from each patient under two different conditions: during anaphylaxis and 14 days after the reaction (control). The sncRNA analysis was carried out by Next Generation Sequencing (NGS). A total of 671 sncRNAs (3 piRNAs, 74 snoRNAs, 54 snRNAs, 348 tRFs and 192 YRFs) were identified in adults with drug-induced anaphylaxis, while 612 sncRNAs (2 piRNAs, 73 snoRNAs, 52 snRNAs, 321 tRFs and 164 YRFs) were characterized in children with food-mediated anaphylaxis. However, only 33 (1 piRNA, 4 snoRNAs, 1 snRNAs, 7 tRFs and 20 YRFs) and 80 (4 snoRNAs, 6 snRNAs, 54 tRFs and 16 YRFs) of them were statistically different between both conditions, respectively. Among them, only three (Y_RNA.394, Y_RNA.781 and SCARNA2) were common to both adults and children analysis. This study provides a differential profile of circulating serum sncRNAs beyond miRNAs in patients with anaphylaxis, postulating them as candidate biomarkers for this pathological event and as novel mediators of the reaction.

Specific Multiomic Profiling in Aortic Stenosis in Bicuspid Aortic Valve Disease.

Bicuspid aortic valve (BAV) disease is associated with faster aortic valve degeneration and a high incidence of aortic stenosis (AS). In this study, we aimed to identify differences in the pathophysiology of AS between BAV and tricuspid aortic valve (TAV) patients in a multiomics study integrating metabolomics and transcriptomics as well as clinical data. Eighteen patients underwent aortic valve replacement due to severe aortic stenosis: 8 of them had a TAV, while 10 of them had a BAV. RNA sequencing (RNA-seq) and proton nuclear magnetic resonance spectroscopy (1H-NMR) were performed on these tissue samples to obtain the RNA profile and lipid and low-molecular-weight metabolites. These results combined with clinical data were posteriorly compared, and a multiomic profile specific to AS in BAV disease was obtained. H-NMR results showed that BAV patients with AS had different metabolic profiles than TAV patients. RNA-seq also showed differential RNA expression between the groups. Functional analysis helped connect this RNA pattern to mitochondrial dysfunction. Integration of RNA-seq, 1H-NMR and clinical data helped create a multiomic profile that suggested that mitochondrial dysfunction and oxidative stress are key players in the pathophysiology of AS in BAV disease. The pathophysiology of AS in BAV disease differs from patients with a TAV and has a specific RNA and metabolic profile. This profile was associated with mitochondrial dysfunction and increased oxidative stress.

3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B-Cell Acute Lymphoblastic Leukemia.

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling.

Quantitative and qualitative subgenomic RNA profiles of SARS-CoV-2 in respiratory samples: A comparison between Omicron BA.2 and non-VOC-D614G

The SARS-CoV-2 Omicron variants are notorious for their transmissibility, but little is known about their subgenomic RNA (sgRNA) expression. This study applied RNA-seq to delineate the quantitative and qualitative profiles of canonical sgRNA of 118 respiratory samples collected from patients infected with Omicron BA.2 and compared with 338 patients infected with non-variant of concern (non-VOC)-D614G. A unique characteristic profile depicted by the relative abundance of 9 canonical sgRNAs was reproduced by both BA.2 and non-VOC-D614G regardless of host gender, age and presence of pneumonia. Remarkably, such profile was lost in samples with low viral load, suggesting a potential application of sgRNA pattern to indicate viral activity of individual patient at a specific time point. A characteristic qualitative profile of canonical sgRNAs was also reproduced by both BA.2 and non-VOC-D614G. The presence of a full set of canonical sgRNAs carried a coherent correlation with crude viral load (AUC ​= ​0.91, 95% CI 0.88–0.94), and sgRNA ORF7b was identified to be the best surrogate marker allowing feasible routine application in characterizing the infection status of individual patient. Further potentials in using sgRNA as a target for vaccine and antiviral development are worth pursuing.

Abstract B059: Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease with an average survival of less than 50%. Although immunotherapy is showing promising results, many patients do not respond, and chemotherapy remains the main treatment. High TNBC recurrence rates are in part due to its molecular heterogeneity. Here, we sought to characterise the sub-clonal and spatial diversity of chemotherapy resistant TNBC to identify the epigenetic hallmarks of lethal sub-populations of cells that may induce cell state transition, therapy resistance and ultimately metastatic progression. Methods: Sixteen primary untreated triple-negative breast tumours (eight chemotherapy sensitive (ChemoS) and eight chemotherapy resistant (ChemoR, who died of their disease within three years)) were subjected to cell-matched high-depth multiome single nuclei RNA- and ATAC-sequencing. Four tumours were subject to spatial RNA and ATAC profiling. Fourteen TNBC PDX models generated from patients with residual disease post NAC were subjected to single nuclei sequencing and used for validation. Meta-clustering of epithelial cells from the RNA profiles was used to identify sub-populations of cells harbouring distinct epigenetic and transcriptomic features enriched in ChemoR patients. Findings were validated in the BrighTNess TNBC NAC clinical trial and in chemotherapy treated TNBC patients from the SCAN-B study. Results: Unsupervised meta-clustering (MC) of epithelial cells in G1 phase of the cell cycle identified nine MC’s across the dataset, including a distinct immune-like MC. Two MCs were dominated by cells from ChemoR patients and were defined by hypoxia, EMT, and extra-cellular matrix signalling. Sub-populations of cells in the ChemoR dominant MCs were underpinned by both promoter and distal differential chromatin accessibility, that mapped to H3K27ac enhancer sites, suggesting a role of enhancer regulation of MC transcription. Spatial ATAC and RNA profiling of a subset of tumours identified spatially epigenetically defined regions that showed distinct chromatin profiles, suggesting that the epigenetic regulation of genes that define the MCs are spatially distinct. ChemoR clusters were independently reproduced in a cohort of fourteen residual disease TNBC PDXs. Genes pertaining to the immune-like MC were associated with a good prognosis in independent cohorts of contemporary treated TNBC (SCAN-B) and with response to NAC in the BrighTNess trial. Marker genes from ChemoR dominant MCs were associated with poor prognosis and significant levels of residual disease following NAC. Conclusions: Our multi-modal integrative analysis reveals unprecedented insight into the role of epigenetically defined spatially distinct, sub-clonal populations that pre-exist in treatment naïve TNBC and predict therapy response. This suggests pre-existing populations of cells have already acquired the epigenetic footprints and hence transcriptomic features to allow them to evade therapy. Citation Format: Fatemeh Ahmadimoghari, Yu Zhang, Ioanna Mavrommatis, Shafali Thakur, Chris Starling, Ioannis Roxanis, Syed Haider, Rachael Natrajan. Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B059.

Profiling of Long Non-Coding RNAs in Auricular Cartilage of Patients with Isolated Microtia.

Introduction: Microtia is the second most common maxillofacial birth defect worldwide. However, the involvement of long non-coding RNAs (lncRNAs) in isolated microtia is not well understood. This study aimed at identifying lncRNAs that regulate the expression of genes associated with isolated microtia. Methods: We used our microarray data to analyze the expression pattern of lncRNA in the auricular cartilage tissues from 10 patients diagnosed with isolated microtia, alongside 15 control subjects. Five lncRNAs were chosen for validation using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: We identified 4651 differentially expressed lncRNAs in the auricular cartilage from patients with isolated microtia. By Gene Ontology/Kyoto Encyclopedia of Genes and Genomes pathway (GO/KEGG) analysis, we identified 27 differentially expressed genes enriched in pathways associated with microtia. In addition, we predicted 9 differentially expressed genes as potential cis-acting targets of 12 differentially expressed lncRNAs. Our findings by qRT-PCR demonstrate significantly elevated expression levels of ZFAS1 and DAB1-AS1, whereas ADIRF-AS1, HOTAIRM1, and EPB41L4A-AS1 exhibited significantly reduced expression levels in the auricular cartilage tissues of patients with isolated microtia. Conclusions: Our study sheds light on the potential involvement of lncRNAs in microtia and provides a basis for further investigation into their functional roles and underlying mechanisms.

LncRNA CHKB-DT Downregulation Enhances Dilated Cardiomyopathy Through ALDH2.

Human cardiac long noncoding RNA (lncRNA) profiles in patients with dilated cardiomyopathy (DCM) were previously analyzed, and the long noncoding RNA CHKB (choline kinase beta) divergent transcript (CHKB-DT) levels were found to be mostly downregulated in the heart. In this study, the function of CHKB-DT in DCM was determined. Long noncoding RNA expression levels in the human heart tissues were measured via quantitative reverse transcription-polymerase chain reaction and in situ hybridization assays. A CHKB-DT heterozygous or homozygous knockout mouse model was generated using the clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, and the adeno-associated virus with a cardiac-specific promoter was used to deliver the RNA in vivo. Sarcomere shortening was performed to assess the primary cardiomyocyte contractility. The Seahorse XF cell mitochondrial stress test was performed to determine the energy metabolism and ATP production. Furthermore, the underlying mechanisms were explored using quantitative proteomics, ribosome profiling, RNA antisense purification assays, mass spectrometry, RNA pull-down, luciferase assay, RNA-fluorescence in situ hybridization, and Western blotting. CHKB-DT levels were remarkably decreased in patients with DCM and mice with transverse aortic constriction-induced heart failure. Heterozygous knockout of CHKB-DT in cardiomyocytes caused cardiac dilation and dysfunction and reduced the contractility of primary cardiomyocytes. Moreover, CHKB-DT heterozygous knockout impaired mitochondrial function and decreased ATP production as well as cardiac energy metabolism. Mechanistically, ALDH2 (aldehyde dehydrogenase 2) was a direct target of CHKB-DT. CHKB-DT physically interacted with the mRNA of ALDH2 and fused in sarcoma (FUS) through the GGUG motif. CHKB-DT knockdown aggravated ALDH2 mRNA degradation and 4-HNE (4-hydroxy-2-nonenal) production, whereas overexpression of CHKB-DT reversed these molecular changes. Furthermore, restoring ALDH2 expression in CHKB-DT+/- mice alleviated cardiac dilation and dysfunction. CHKB-DT is significantly downregulated in DCM. CHKB-DT acts as an energy metabolism-associated long noncoding RNA and represents a promising therapeutic target against DCM.

Robust detection of clinically relevant features in single-cell RNA profiles of patient-matched fresh and formalin-fixed paraffin-embedded (FFPE) lung cancer tissue

PurposeSingle-cell transcriptional profiling reveals cell heterogeneity and clinically relevant traits in intra-operatively collected patient-derived tissue. So far, single-cell studies have been constrained by the requirement for prospectively collected fresh or cryopreserved tissue. This limitation might be overcome by recent technical developments enabling single-cell analysis of FFPE tissue.MethodsWe benchmark single-cell profiles from patient-matched fresh, cryopreserved and archival FFPE cancer tissue.ResultsWe find that fresh tissue and FFPE routine blocks can be employed for the robust detection of clinically relevant traits on the single-cell level. Specifically, single-cell maps of fresh patient tissues and corresponding FFPE tissue blocks could be integrated into common low-dimensional representations, and cell subtype clusters showed highly correlated transcriptional strengths of signaling pathway, hallmark, and clinically useful signatures, although expression of single genes varied due to technological differences. FFPE tissue blocks revealed higher cell diversity compared to fresh tissue. In contrast, single-cell profiling of cryopreserved tissue was prone to artifacts in the clinical setting.ConclusionOur analysis highlights the potential of single-cell profiling in the analysis of retrospectively and prospectively collected archival pathology cohorts and increases the applicability in translational research.

Whole-Transcriptome Analysis Reveals the RNA Profiles in Mouse Bone Marrow Mesenchymal Stem Cells or Zebrafish Embryos After Exposure to Environmental Level of Tributyltin.

To understand the underlying molecular mechanisms, mouse bone marrow mesenchymal stem cells (BMSCs) and zebrafish embryos were exposed to the control group and Tributyltin (TBT) group (10 ng/L, environmental concentration) for 48h, respectively. The expression profiles of RNAs were investigated using whole-transcriptome analysis in mouse BMSCs or zebrafish embryos after TBT exposure. For mouse BMSCs, the results showed 2,449 differentially expressed (DE) mRNAs, 59 DE miRNAs, 317 DE lncRNAs, and 15 circRNAs. Similarly, for zebrafish embryos, the results showed 1,511 DE mRNAs, 4 DE miRNAs, 272 DE lncRNAs, and 28 circRNAs. According to KEGG pathway analysis showed that DE RNAs were mainly associated with immune responses, signaling, and cellular interactions. Competing endogenous RNA (ceRNA) network analysis revealed that the regulatory network of miRNA-circRNA constructed in zebrafish embryos was more complex compared to that of mouse BMSCs.

Transcriptome-wide N6-methyladenosine (m6A) methylation profiling of long non-coding RNAs in ovarian endometriosis

N6-methyladenosine (m6A) methylation is the most prevalent internal epigenetic posttranscriptional mechanism for regulating mammalian RNA. Despite recent advances in determining the biological functions of m6A methylation, its association with the pathology of ovarian endometriosis remains uncertain. Herein, we performed m6A transcriptome-wide profiling to identify key lncRNAs with m6A modification involved in ovarian endometriosis development by bioinformatics analysis. We found the total m6A level was lower in ovarian endometriosis than in normal endometrium samples, with 9663 m6A peaks associated with 8989 lncRNAs detected in ovarian endometriosis and 9902 m6A peaks associated with 9210 lncRNAs detected in normal endometrium samples. These m6A peaks were primarily enriched within AAACU motifs. Functional enrichment analysis indicated that pathways involving the regulation of adhesion and development were significantly enriched in these differentially methylated lncRNAs. The regulatory relationships among lncRNAs, microRNAs (miRNAs), and mRNAs were identified by competing endogenous RNA (ceRNA) analysis and determination of the network regulating lncRNA-mRNA expression. Several specific lncRNA, including LINC00665, LINC00937, FZD10-AS1, DIO3OS and GATA2-AS1 which were differently expressed and modified by m6A, were validated using qRT-PCR and its interaction with infiltrating immune cells was explored. Furthermore, we found LncRNA DIO3OS promotes the invasion and migration of Human endometrial stromal cells (THESCs) and ALKBH5 regulates the expression of the lncRNA DIO3OS through m6A modification in vitro. Our study firstly revealed the transcriptome-wide map of m6A modification in lncRNAs of ovarian endometriosis. These findings may enable the determination of the underlying mechanism governing the pathogenesis of ovarian endometriosis and provide theoretical basis for further deeper research on the role of m6A in the development of ovarian endometriosis.

Pivotal role for long noncoding RNAs in zygotic genome activation in mice.

Vertebrate life begins with fertilization, and then the zygote genome is activated after transient silencing, a process termed zygotic genome activation (ZGA). Despite its fundamental role in totipotency and the initiation of life, the precise mechanism underlying ZGA initiation remains unclear. The existence of minor ZGA implies the possible critical role of noncoding RNAs in the initiation of ZGA. Here, we delineate the expression profile of long noncoding RNAs (lncRNAs) in early mouse embryonic development and elucidate their critical role in minor ZGA. Compared with protein-coding genes (PCGs), lncRNAs exhibit a stronger correlation with minor ZGA. Distinct H3K9me3 profiles can be observed between lncRNA genes and PCGs, and the enrichment of H3K9me3 before ZGA might explain the suspended expression of major ZGA-related PCGs despite possessing PolII pre-configuration. Furthermore, we identified the presence of PolII-enriched MuERV-L around the transcriptional start site of minor ZGA-related lncRNAs, and these repeats are responsible for the activation of minor ZGA-related lncRNAs and subsequent embryo development. Our study suggests that MuERV-L mediates minor ZGA lncRNA activation as a critical driver between epigenetic reprogramming triggered by fertilization and the embryo developmental program, thus providing clues for understanding the regulatory mechanism of totipotency and establishing bona fide totipotent stem cells.

Differential expression of tsRNAs and miRNAs in embryo culture medium: potential impact on embryo implantation.

Can small RNA derived from embryos in conditioned embryo culture medium (ECM) influence embryo implantation? We employed small RNA sequencing to investigate the expression profiles of transfer RNA-derived small RNA (tsRNA) and microRNA (miRNA) in ECM from high-quality and low-quality embryos. Quantitative real-time PCR was employed to validate the findings of small RNA sequencing. Additionally, we conducted bioinformatics analysis to predict the potential functions of these small RNAs in embryo implantation. To establish the role of tiRNA-1:35-Leu-TAG-2 in embryonic trophoblast cell adhesion, we utilized co-culture systems involving JAR and Ishikawa cells. Our analysis revealed upregulation of nine tsRNAs and four miRNAs in ECM derived from high-quality embryos, whereas 37 tsRNAs and 12 miRNAs exhibited upregulation in ECM from low-quality embryos. The bioinformatics analysis of tsRNA, miRNA, and mRNA pathways indicated that their respective target genes may play pivotal roles in both embryo development and endometrial receptivity. Utilizing tiRNA mimics, we demonstrated that the prominently expressed tiRNA-1:35-Leu-TAG-2 in the low-quality ECM group can be internalized by Ishikawa cells. Notably, transfection of tiRNA-1:35-Leu-TAG-2 into Ishikawa cells reduced the attachment rate of JAR spheroids. Our investigation uncovers significant variation in the expression profiles of tsRNAs and miRNAs between ECM derived from high- and low-quality embryos. Intriguingly, the release of tiRNA-1:35-Leu-TAG-2 by low-quality embryos detrimentally affects embryo implantation and endometrial receptivity. These findings provide fresh insights into understanding the molecular foundations of embryo-endometrial communication.

Consistency between Primary Uterine Corpus Malignancies and Their Corresponding Patient-Derived Xenograft Models.

Patient-derived xenograft (PDX) models retain the characteristics of tumors and are useful tools for personalized therapy and translational research. In this study, we aimed to establish PDX models for uterine corpus malignancies (UC-PDX) and analyze their similarities. Tissue fragments obtained from 92 patients with uterine corpus malignancies were transplanted subcutaneously into immunodeficient mice. Histological and immunohistochemical analyses were performed to compare tumors of patients with PDX tumors. DNA and RNA sequencing were performed to validate the genetic profile. Furthermore, the RNA in extracellular vesicles (EVs) extracted from primary and PDX tumors was analyzed. Among the 92 cases, 52 UC-PDX models were established, with a success rate of 56.5%. The success rate depended on tumor histology and staging. The pathological and immunohistochemical features of primary and PDX tumors were similar. DNA sequencing revealed similarities in gene mutations between the primary and PDX tumors. RNA sequencing showed similarities in gene expressions between primary and PDX tumors. Furthermore, the RNA profiles of the EVs obtained from primary and PDX tumors were similar. As UC-PDX retained the pathological and immunohistochemical features and gene profiles of primary tumors, they may provide a platform for developing personalized medicine and translational research.

RNA Profile of Cell Bodies and Exosomes Released by Tumorigenic and Non-Tumorigenic Thyroid Cells.

Tumor cells release exosomes, extracellular vesicle containing various bioactive molecules such as protein, DNA and RNA. The analysis of RNA molecules packaged in exosomes may provide new potential diagnostic or prognostic tumor biomarkers. The treatment of radioiodine-refractory aggressive thyroid cancer is still an unresolved clinical challenge, and the search for biomarkers that are detectable in early phase of the disease has become a fundamental goal for thyroid cancer research. By using transcriptome analysis, this study aimed to analyze the gene expression profiles of exosomes secreted by a non-tumorigenic thyroid cell line (Nthy-ori 3.1-exo) and a papillary thyroid cancer (TPC-1-exo) cell line, comparing them with those of cell bodies (Nthy-ori 3.1-cells and TPC-1-cells). A total of 9107 transcripts were identified as differentially expressed when comparing TPC-1-exo with TPC-1-cells and 5861 when comparing Nthy-ori 3.1-exo with Nthy-ori 3.1-cells. Among them, Sialic acid-binding immunoglobulin-like lectins 10 and 11 (SIGLEC10, SIGLEC11) and Keratin-associated protein 5 (KRTAP5-3) transcripts, genes known to be involved in cancer progression, turned out to be up-regulated only in TPC-1-exo. Gene ontology analysis revealed significantly enriched pathways, and only in TPC-1-exo were the differential expressed genes associated with an up-regulation in epigenetic processes. These findings provide a proof of concept that some mRNA species are specifically packaged in tumor-cell-derived exosomes and may constitute a starting point for the identification of new biomarkers for thyroid tumors.

Loratadine as an Anti-inflammatory Agent Against Clostridium difficile Toxin B.

Clostridium difficile infection (CDI) is a debilitating nosocomial infection. C. difficile produces toxins A and B, which cause inflammation. Existing therapies have issues with recurrence, cost, and safety. We aim to discover a safe, effective, and economical nonmicrobiological therapeutic approach against CDI. We included human primary peripheral blood mononuclear cells (PBMCs), fresh human colonic explants, and humanized HuCD34-NCG mice. Toxin A+B+ VPI 10463 and A-B+ ribotype 017 C. difficile strains were used. We used single-cell RNA profiling and high-throughput screening to find actionable toxin B-dependent pathways in PBMCs. Histamine 1 receptor-related drugs were found among the hit compounds that reversed toxin-mediated macrophage inflammatory protein (MIP) 1α expression in PBMCs. We identified loratadine as the safest representative antihistamine for therapeutic development. Loratadine inhibited toxin B-induced MIP-1α secretion in fresh human colonic tissues. Oral loratadine (10 mg/kg/d) maintained survival, inhibited intestinal CCl3 messenger RNA expression, and prevented vancomycin-associated recurrence in the VPI 10463-infected mice and ribotype 017-infected hamsters. Splenocytes from loratadine-treated mice conferred anti-inflammatory effects to the VPI 10463-infected T/B-cell--deficient Rag-/- mice. Oral loratadine suppressed human MIP-1α expression in monocytes/macrophages in toxin B-expressing ribotype 017-infected humanized HuCD34-NCG mice. Loratadine may be repurposed to optimize existing therapies against CDI.

Super enhancer loci of EGFR regulate EGFR variant 8 through enhancer RNA and strongly associate with survival in HNSCCs.

Epidermal growth factor receptor (EGFR) has been shown to be overexpressed in human cancers due to mutation, amplification, and epigenetic hyperactivity, which leads to deregulated transcriptional mechanism. Among the eight different EGFR isoforms, the mechanism of regulation of full-length variant 1 is well-known, no studies have examined the function & factors regulating the expression of variant 8. This study aimed to understand the function of EGFR super-enhancer loci and its associated transcription factors regulating the expression of EGFR variant 8. Our study shows that overexpression of variant 8 and its transcription was more prevalent than variant 1 in many cancers and positively correlated with the EGFR-AS1 expression in oral cancer and HNSCC. Notably, individuals overexpressing variant 8 showed shorter overall survival and had a greater connection with other clinical traits than patients with overexpression of variant 1. In this study, TCGA enhancer RNA profiling on the constituent enhancer (CE1 and CE2) region revealed that the multiple enhancer RNAs formed from CE2 by employing CE1 as a promoter. Our bioinformatic analysis further supports the enrichment of enhancer RNA specific chromatin marks H3K27ac, H3K4me1, POL2 and H2AZ on CE2. GeneHancer and 3D chromatin capture analysis showed clustered interactions between CE1, CE2 loci and this interaction may regulates expression of both EGFR-eRNA and variant 8. Moreover, increased expression of SNAI2 and its close relationship to EGFR-AS1 and variant 8 suggest that SNAI2 could regulates variant 8 overexpression by building a MegaTrans complex with both EGFR-eRNA and EGFR-AS1. Our findings show that EGFR variant 8 and its transcriptional regulation & chromatin modification by eRNAs may provide a rationale for targeting RNA splicing in combination with targeted EGFR therapies in cancer.

Novel Insights into the Circadian Rhythms Based on Long Noncoding and Circular RNA Profiling.

Circadian rhythm disorders pose major risks to human health and animal production activity, and the hypothalamus is the center of circadian rhythm regulation. However, the epigenetic regulation of circadian rhythm based on farm animal models has been poorly investigated. We collected chicken hypothalamus samples at seven time points in one light/dark cycle and performed long noncoding RNA (lncRNA), circular RNA (circRNA), and mRNA sequencing to detect biomarkers associated with circadian rhythm. We enhanced the comprehensive expression profiling of ncRNAs and mRNAs in the hypothalamus and found two gene sets (circadian rhythm and retinal metabolism) associated with the light/dark cycle. Noncoding RNA networks with circadian expression patterns were identified by differential expression and circadian analysis was provided that included 38 lncRNAs, 15 circRNAs, and 200 candidate genes. Three lncRNAs (ENSGALT00000098661, ENSGALT00000100816, and MSTRG.16980.1) and one circRNA (novel_circ_010168) in the ncRNA-mRNA regulatory network were identified as key molecules influencing circadian rhythm by regulating AOX1 in retinal metabolism. These ncRNAs were predicted to be related to pernicious anemia, gonadal, eye disease and other disorders in humans. Together, the findings of this study provide insights into the epigenetic mechanisms of circadian rhythm and reveal AOX1 as a promising target of circadian rhythm regulation.