Whole-transcriptome RNA Sequencing Research Articles

Abstract GS03-07: Protocol-defined biomarker analysis in the PALLAS (AFT-05) adjuvant trial: Genomic subtype derived from RNA sequencing of HR+/HER2- early breast cancer

Abstract Background. The phase 3 PALLAS trial (NCT02513394) compared two years of the CDK4/6 inhibitor palbociclib with endocrine therapy of provider choice, versus endocrine therapy alone, as adjuvant treatment for patients with Stage II-III hormone receptor-positive HER2-negative (HR+/HER2-) breast cancer. Genomic subtype (PAM50 intrinsic subtype) measured from whole-transcriptome RNA sequencing data was defined in the protocol of the PALLAS trial as the primary biomarker for analysis of prediction and prognosis. Clinical data have been previously presented (Gnant et al, JCO 2022), and the trial now has 5-year median follow-up. Methods. As part of trial eligibility, all participants in PALLAS provided a tumor tissue block prior to randomization (surgical if primary resection, core biopsy if neoadjuvant treatment) for translational analyses (TRANS-PALLAS). The biorepository and laboratory were blinded to identity and processed samples in random order, to minimize bias. Nucleic acids were extracted from samples with sufficient tumor tissue and cellularity (>25 mm2 with ≥20% cancer nuclei). The Genome Sequencing Center at Washington University St. Louis performed whole-transcriptome RNA sequencing. Libraries were prepared from 1 µg DNase-1 treated total RNA, if total RNA DV200 >28 (Agilent Bioanalyzer), using an unbiased library protocol of RNA HyperPrep kit with RiboErase (HMR) (Kapa Biosystems, Wilmington, MA). 100 bp paired-end sequencing was performed on NovaSeq 6000 using S4 Reagent Kit (Illumina, San Diego, CA), with 48 libraries pooled per lane. Intrinsic subtype was determined using Bioclassifier package (Research PAM50 script, Parker et al.) only for the analysis population of primary breast cancer samples that had not been exposed to prior neoadjuvant therapy. Invasive disease-free survival (IDFS) will be visualized using Kaplan-Meier plots, with log-rank test between groups. Cox models of proportional hazards will be developed to evaluate prognosis adjusted for known clinical covariates, or for predictive interactions. The pre-defined level of significance is a two-sided 0.05. Results. From the total study population of 5796 enrolled patients, 4655 tissue blocks had sufficient tumor content to process for RNA, with 3931 yielding sufficient RNA for sequencing, and 2669/4655 (57.3%) submitted tissue blocks had DV200 ≥28 and were successfully sequenced. Clinical unblinding revealed 2370 unique patients (1182 in the palbociclib treatment arm and 1188 in the control arm) with intrinsic subtype defined from their untreated primary tumor: 1555 (65.6%) luminal A, 287 (12.1%) luminal B, 167 (7.0%) HER2-enriched, 310 (13.1%) basal-like, 51 (2.2%) normal-like. We will report the results for association of molecular subtype, proliferation score, and Risk of Recurrence (ROR) scores with invasive disease-free survival (IDFS) by treatment arm at the meeting. Conclusions. The TRANS-PALLAS cohort represents one of the largest biorepositories of HR+/HER2- early breast cancer reflecting contemporary systemic management in the framework of a prospectively randomized global trial. Required tumor block submission in this phase 3 trial yielded data from unbiased whole-transcriptome RNA sequencing of the primary tumor prior to treatment from 41% of the PALLAS participants. The proportion of luminal A cancers was unexpectedly high (66%), indicating a lower-risk distribution of cancers in this population. The planned analyses of prediction and prognosis are ongoing and those results will be presented at the time of the meeting. Support: AFT, ABCSG, Pfizer, ECOG-ACRIN, NSABP Foundation, GBG, BIG; Clinicaltrials.gov Identifier: NCT02513394; https://acknowledgments.alliancefound.org Citation Format: Daniel Stover, Dominik Hlauschek, Erica Mayer, W. Fraser Symmans, Mark Watson, Iros Barozzi, Martin Filipits, Karla Ballman, Meritxell Bellet- Ezquerra, Justin Balko, Gabor Rubovszky, Nicholas Zdenkowski, Adam Brufsky, Guenther Steger, Claudine Isaacs, Sibylle Loibl, Fernando Henao, Meredith Regan, Yuan Liu, Christian Fesl, Patrick O'Brien, Angela DeMichele, Michael Gnant, Otto Metzger. Protocol-defined biomarker analysis in the PALLAS (AFT-05) adjuvant trial: Genomic subtype derived from RNA sequencing of HR+/HER2- early breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-07.

Abstract 4898: Highly accurate profiling of leukocyte composition from bulk peripheral blood with targeted digital cytometry

Abstract Background: Cell profiling methods such as flow/mass cytometry and single-cell RNA-sequencing are powerful tools for quantifying immune composition from healthy and neoplastic tissues. However, only a modest number of markers can be interrogated by the former and the latter remains cost prohibitive for large-scale analysis. Here we demonstrate robust, accurate, and reproducible enumeration of immune cell subsets from 36 whole blood samples using SureSelect XT HS2 RNA sequencing combined with CiberMed’s iSortTM digital cytometry solution. Enrichment of genes in the LM22 signature matrix, a well-established collection of reference profiles for deconvolving 22 human immune subsets, was achieved with two new targeted sequencing panels—Agilent SureSelect CD CiberMed Heme and Agilent SureSelect CD CiberMed Heme + HiRes. Both panels were assessed for their ability to profile leukocyte subsets with CiberMed’s iSortTM Fractions software, an optimized and standardized version of CIBERSORTx with novel enhancements. Methods: Whole blood samples were freshly collected from 36 healthy donors and split into two fractions—one was immediately processed for complete blood count (CBC) and flow cytometric enumeration of major leukocyte populations; the other was stored in PAXgene Blood RNA tubes for subsequent RNA sequencing. A Sysmex system was employed for CBC quantification and a Becton Dickinson 6-color TBNK MultiTest in vitro diagnostic (IVD) assay was employed for enumerating B cells, CD8 T cells, CD4 T cells, NK cells. These data were used as ground truth to assess iSortTM deconvolution performance from targeted and whole-transcriptome bulk RNA sequencing data. RNA-seq expression values were used as input to iSortTM Fractions to deconvolve 22 immune subsets in each sample. Results: Across 7 major populations, cell type fractions determined by iSortTM Fractions were highly concordant with ground truth fractions determined by clinical grade standards (r≥0.96) and exhibited strong reproducibility across technical replicates (r≥0.98). Furthermore, targeted enrichment using the SureSelect CD CiberMed Heme panel reduced the sequencing requirement by nearly 50-fold compared to whole-transcriptome sequencing, while also improving accuracy. Conclusion: These new panels for digital cytometry are being released through the Agilent Community Design program to enable focused, reliable, and high-throughput analysis of cell type composition from peripheral blood samples. Citation Format: Aki Nakao, Aaron M. Newman, Ash A. Alizadeh, Maximilian Diehn, Mistuni Ghosh, Manjula Aliminati, Jayati Ghosh, Kristi Stephenson, Ashutosh Ashutosh. Highly accurate profiling of leukocyte composition from bulk peripheral blood with targeted digital cytometry [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 4898.

RNA-sequencing of paired tape-strips and skin biopsies in atopic dermatitis reveals key differences.

Skin tape-strips and biopsies are widely used methods for investigating the skin in atopic dermatitis (AD). Biopsies are more commonly used but can cause scarring and pain, whereas tape-strips are noninvasive but sample less tissue. The study evaluated the performance of skin tape-strips and biopsies for studying AD. Whole-transcriptome RNA-sequencing was performed on paired tape-strips and biopsies collected from lesional and non-lesional skin from AD patients (n = 7) and non-AD controls (n = 5). RNA yield, mapping efficiency, and differentially expressed genes (DEGs) for the two methods (tape-strip/biopsy) and presence of AD (AD/non-AD) were compared. Tape-strips demonstrated a lower RNA yield (22 vs. 4596 ng) and mapping efficiency to known genes (28% vs. 93%) than biopsies. Gene-expression profiles of paired tape-strips and biopsies demonstrated a medium correlation (R2 = 0.431). Tape-strips and biopsies demonstrated systematic differences in measured expression levels of 6483 genes across both AD and non-AD samples. Tape-strips preferentially detected many itch (CCL3/CCL4/OSM) and immune-response (CXCL8/IL4/IL5/IL22) genes as well as markers of epidermal dendritic cells (CD1a/CD207), while certain cytokines (IL18/IL37), skin-barrier genes (KRT2/FLG2), and dermal fibroblasts markers (COL1A/COL3A) were preferentially detected by biopsies. Tape-strips identified more DEGs between AD and non-AD (3157 DEGs) then biopsies (44 DEGs). Tape-strips also detected higher levels of bacterial mRNA than biopsies. This study concludes that tape-strips and biopsies each demonstrate respective advantages for measuring gene-expression changes in AD. Thus, the specific skin layers and genes of interest should be considered before selecting either method.

Abstract B068: FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models

Abstract The unfavorable prognosis of high-grade serous ovarian cancer, HGSOC, may be driven by a small subset of ovarian cancer stem cells (OCSC), which are endowed with tumor-initiating ability and are intrinsically resistant to cytotoxic treatments. These characteristics are sustained by signals coming from the tumor microenvironment (TME). As a common site of HGSOC presentation is the omentum, we partially recapitulated the architecture of human omentum in vitro using 3D co-cultures of primary human omental fibroblasts and mesothelial cells. Patient-derived HGSOC cells were then integrated in these organotypic models, either as bulk cancer cells or as OCSC from the same patients. After co-culture in this model, malignant cells were re-isolated by FACS sorting and subjected to whole-transcriptome RNA sequencing, comparing them to control cells that have not been in contact with the in vitro TME. Our data revealed transcriptional reprogramming induced by the in vitro TME in HGSOC cells. We found a number of pathways differentially regulated in the bulk population of OC cells vs OCSC, shedding light on the impact of the TME on OC stemness. Moreover, we identified a panel of transcription factors (TFs) specifically altered by the in vitro TME in OCSC. These TF-regulated gene networks represent OCSC vulnerabilities and, hence, potential targets in relapsing tumors. Among these, we focused on FOXM1, a TF with pleiotropic functions in cancer cells, whose activity is altered in 84% of ovarian cancer patients. The FOXM1 pathway was strongly induced by the in vitro TME specifically in OCSC. Mechanistically, the contact with the in vitro TME triggered a FAK/YAP axis which, in turn, accounted for the induction of FOXM1 and its downstream target genes. Moreover, the pharmacological inhibition of FOXM1 significantly impaired OCSC fitness, supporting, on one hand, the key role of TME in orchestrating OC stemness and suggesting, on the other, a new approach for the eradication of OCSC from the omental niche. Overall, our patient-derived organotypic platform offers the opportunity to unravel the impact of the TME on OCSC at the molecular level in experimental models with clinical relevance and can be exploited to design innovative HGSOC-eradicating treatments. Citation Format: Chiara Battistini, Hilary A. Kenny, Valentina Nieddu, Valentina Melocchi, Alessandra Decio, Beatrice Malacrida, Alessia Gatto, Fabrizio Bianchi, Raffaella Giavazzi, Nicoletta Colombo, Frances R. Balkwill, Ernst Lengyel, Ugo Cavallaro. FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B068.

Whole-transcriptome RNA sequencing reveals CeRNA regulatory network under long-term space composite stress in Rats

To systematically evaluate the effect of simulated long-term spaceflight composite stress (LSCS) in hippocampus and gain more insights into the transcriptomic landscape and molecular mechanism, we performed whole-transcriptome sequencing based on the control group (Ctrl) and the simulated long-term spaceflight composite stress group (LSCS) from six hippocampus of rats. Subsequently, differential expression analysis was performed on the Ctrl and LSCS groups, followed by enrichment analysis and functional interaction prediction analysis to investigate gene-regulatory circuits in LSCS. In addition, competitive endogenous RNA (ceRNA) network was constructed to gain insights into genetic interaction. The result showed that 276 differentially expressed messenger RNAs (DEmRNAs), 139 differentially expressed long non-coding RNAs (DElncRNAs), 103 differentially expressed circular RNAs (DEcircRNAs), and 52 differentially expressed microRNAs (DEmiRNAs) were found in LSCS samples compared with the controls, which were then subjected to enrichment analysis of Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways to find potential functions. PI3K-Akt signaling pathway and MAPK signaling pathway may play fundamental roles in the pathogenesis of LSCS. A ceRNA network was constructed with the predicted 340 DE pairs, which revealed the interaction roles of 220 DEmiRNA-DEmRNA pairs, 76 DEmiRNA-DElncRNA pairs, and 44 DEmiRNA-DEcircRNA pairs. Further, Thrombospondins2 was found to be a key target among those ceRNAs. Overall, we conducted for the first time a full transcriptomic analysis of the response of hippocampus to the LSCS that involved a potential ceRNA network, thus providing a basis to study the underlying mechanism of the LSCS.

Noncoding RNA-associated competing endogenous RNA networks in trastuzumab-induced cardiotoxicity

Trastuzumab-induced cardiotoxicity (TIC) is a common and serious disease with abnormal cardiac function. Accumulating evidence has indicated certain non-coding RNAs (ncRNAs), functioning as competing endogenous RNAs (ceRNAs), impacting the progression of cardiovascular diseases. Nonetheless, the specific involvement of ncRNA-mediated ceRNA regulatory mechanisms in TIC remains elusive. The present research aims to comprehensively investigate changes in the expressions of all ncRNA using whole-transcriptome RNA sequencing. The sequencing analysis unveiled significant dysregulation, identifying a total of 43 circular RNAs (circRNAs), 270 long noncoding RNAs (lncRNAs), 12 microRNAs (miRNAs), and 4131 mRNAs in trastuzumab-treated mouse hearts. Subsequently, circRNA-based ceRNA networks consisting of 82 nodes and 91 edges, as well as lncRNA-based ceRNA networks comprising 111 nodes and 112 edges, were constructed. Using the CytoNCA plugin, pivotal genes—miR-31-5p and miR-644-5p—were identified within these networks, exhibiting potential relevance in TIC treatment. Additionally, KEGG and GO analyses were conducted to explore the functional pathways associated with the genes within the ceRNA networks. The outcomes of the predicted ceRNAs and bioinformatics analyses elucidated the plausible involvement of ncRNAs in TIC pathogenesis. This insight contributes to a better understanding of underlying mechanisms and aids in identifying promising targets for effective prevention and treatment strategies.

Oncogenic fusions in renal cell carcinoma.

475 Background: Detecting actionable or diagnostic fusions in solid tumors may significantly alter clinical decisions, including therapy selection. Some fusions have matched drugs approved for use in the patient’s cancer, or in another cancer. Others have published evidence of benefit with particular drugs but are not included in guidelines, and some may represent an inclusion criterion for clinical trials. While diagnostic fusions may not have associated therapies, they are cancer-defining and can lead to re-classification of tumors. Here we describe actionable and diagnostic fusions we have observed in renal cell carcinoma (RCC). Methods: Tumor samples from patients diagnosed with RCC were analyzed using the OncoExTra assay, which identifies somatic mutations and gene fusions through tumor-normal, whole-exome DNA and whole-transcriptome RNA sequencing. All actionable fusions, defined as those with associated FDA-approved targeted therapies in any cancer type, those that made patients eligible for an active clinical trial, or those with evidence in guidelines or the literature for possible matched therapies in any cancer, are detectable using this assay. We examined samples from patients with RCC who had the OncoExtra assay performed between May 2018 and Mar 2023 and identified both actionable and diagnostic fusions. Results: Among 389 tumor samples from patients diagnosed with RCC, there were 11 (2.8%) actionable and 1 (0.3%) diagnostic fusion identified. The actionable fusions included 6 (1.5%) TFE3, 1 (0.3%) ALK, 1 (0.3%) EGFR, 1 (0.3%) ERBB4, 1 (0.3%) YAP and 1 (0.3%) FGFR2, and the diagnostic fusion was NAB2/STAT6. The 6 TFE3 oncogene fusions involved 4 different partner genes ( PRCC was the partner in 3 fusions). ALK, EGFR, ERBB and FGFR fusions are found across a variety of cancers, and all have matched FDA-approved drug therapies in at least one cancer type. For example, ALK fusions, including the observed EML4/ALK fusion, which is common in non-small cell lung cancer, have several matched ALK-inhibitor therapies. TFE3 (transcription factor binding to IGHM enhancer 3) gene fusions are a diagnostic genomic alteration in microphthalmia-associated transcription factor (MiT) translocation RCC, which is a rare but aggressive subtype of RCC. While TFE3 fusions do not have FDA-approved matched therapies in any cancer, they are known to result in PIK3K/AKT/mTOR and MET activation, suggesting inhibitors of these pathways may be considered in these patients. The NAB2/STAT6 diagnostic fusion is characteristic of solitary fibrous tumors, suggesting that the patient likely had this kind of tumor. Conclusions: Whole-transcriptome sequencing allowed the identification of actionable fusions with the potential to affect clinical decisions regarding therapy in 2.8% of RCC patient tumors. In addition, one patient was found to have a fusion that is characteristic of a solitary fibrous tumor, demonstrating the power of genomic profiling to aid diagnosis.

An Immune Gene Expression Risk Score for Distant Metastases after Radiotherapy for Cervical Cancer.

To develop an immune-based gene expression risk score to identify patients with cervical cancer at increased risk of distant metastases (DM). Tumor biopsies were obtained from 81 patients prior to chemoradiotherapy. Whole-transcriptome RNA sequencing was performed (Illumina NextSeq500). Beginning with 4,723 immune-related genes, a 55-gene risk score for DM was derived using Cox modeling and principal component analysis. It was validated in independent cohorts of 274 patients treated at the Norwegian Radium Hospital (NRH) and 206 patients from The Cancer Genome Atlas (TCGA). The risk score was predictive of DM (HR, 2.7; P < 0.0001) and lower cause-specific survival (CSS) by univariate analysis (HR, 2.0; P = 0.0003) and multivariate analysis adjusted for clinical factors (DM HR, 3.0; P < 0.0001; CSS HR, 2.2; P = 0.0004). The risk score predicted DM (HR, 1.4; P = 0.05) and CSS (HR, 1.48; P = 0.013) in the NRH cohort and CSS (HR, 1.4; P = 0.03) in TCGA cohort. Higher risk scores were associated with lower CIBERSORT estimates of tumor-infiltrating immune cells, including CD8 T cells and M1 and M2 macrophages (all P < 0.001). Higher risk scores were associated with lower expression (all P < 0.001) of important chemokines (CXCL12, CXCR4), IFN-regulated genes (IRF1, STAT1, IDO1), and immune checkpoint regulators (PD-1, PD-L1, CTLA-4). The immune metastatic risk score addresses important challenges in the treatment of cervical cancer-identifying patients at high risk of DM after radiotherapy. The findings of this study indicate that high tumor mutational burden and a "cold," immune-excluded tumor microenvironment influence distant metastatic recurrence. Further validation of the risk score is needed.

Identification of ceRNA-vsiRNA-mRNA network for exploring the mechanism underlying pathogenesis of sugarcane mosaic virus in resistant and susceptible maize inbred lines

RNA silencing plays an important role in plant antiviral responses, which trigger the production of virus-derived small interfering RNAs (vsiRNAs). The competing endogenous RNA (ceRNA) hypothesis revealed a unique mechanism in which circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) can interact with small RNAs to regulate the expression of corresponding target mRNAs. Sugarcane mosaic virus (SCMV) infection causes severe economic losses in maize (Zea mays L.) production worldwide. This study compared and analyzed characteristics of vsiRNAs derived from SCMV and their target genes in resistant (Chang7-2) and susceptible (Mo17) maize inbred lines through whole-transcriptome RNA sequencing and degradome sequencing. The results showed that 706 transcripts were targeted by 204 vsiRNAs, including 784 vsiRNA-target gene pairs. Furthermore, ceRNA networks of circRNA/lncRNA-vsiRNA-mRNA in response of maize to SCMV infection were obtained, including 3 differentially expressed (DE) circRNAs, 36 DElncRNAs, 105 vsiRNAs, and 342 DEmRNAs in Mo17 plants, and 3 DEcircRNAs, 35 DElncRNAs, 23 vsiRNAs, and 87 DEmRNAs in Chang7-2 plants. Our results also showed that the transcripts of ZmDCLs, ZmAGOs, and ZmRDRs were differentially accumulated in resistant and susceptible maize inbred lines after SCMV infection. These findings provide valuable insights into the relationship between SCMV-derived vsiRNAs and potential ceRNAs fine-tuning the SCMV-maize interaction and offer novel clues to reveal the mechanism underlying the pathogenesis of SCMV.

Whole-transcriptome characterization and functional analysis of lncRNA-miRNA-mRNA regulatory networks responsive to sugarcane mosaic virus in maize resistant and susceptible inbred lines

Sugarcane mosaic virus (SCMV) is one of the most important pathogens causing maize dwarf mosaic disease, which seriously affects the yield and quality of maize. Currently, the molecular mechanism of non-coding RNAs (ncRNAs) responding to SCMV infection in maize is still uncovered. In this study, a total of 112 differentially expressed (DE)-long non-coding RNAs (lncRNAs), 24 DE-microRNAs (miRNAs), and 1822 DE-messenger RNAs (mRNAs), and 363 DE-lncRNAs, 230 DE-miRNAs, and 4376 DE-mRNAs were identified in maize resistant (Chang7-2) and susceptible (Mo17) inbred lines in response to SCMV infection through whole-transcriptome RNA sequencing, respectively. Moreover, 4874 mRNAs potentially targeted by 635 miRNAs were obtained by degradome sequencing. Subsequently, several crucial SCMV-responsive lncRNA-miRNA-mRNA networks were established, of which the expression levels of lncRNA10865-miR166j-3p-HDZ25/69 (class III homeodomain-leucine zipper 25/69) module, and lncRNA14234-miR394a-5p-SPL11 (squamosal promoter-binding protein-like 11) module were further verified. Additionally, silencing lncRNA10865 increased the accumulations of SCMV and miR166j-3p, while silencing lncRNA14234 decreased the accumulations of SCMV and SPL11 targeted by miR394a-5p. This study revealed the interactions of lncRNAs, miRNAs and mRNAs in maize resistant and susceptible materials, providing novel clues to reveal the mechanism of maize in resistance to SCMV from the perspective of competing endogenous RNA (ceRNA) regulatory networks.

Whole-Transcriptome RNA Sequencing Uncovers the Global Expression Changes and RNA Regulatory Networks in Duck Embryonic Myogenesis.

Duck meat is pivotal in providing high-quality protein for human nutrition, underscoring the importance of studying duck myogenesis. The regulatory mechanisms governing duck myogenesis involve both coding and non-coding RNAs, yet their specific expression patterns and molecular mechanisms remain elusive. To address this knowledge gap, we performed expression profiling analyses of mRNAs, lncRNAs, circRNAs, and miRNAs involved in duck myogenesis using whole-transcriptome RNA-seq. Our analysis identified 1733 differentially expressed (DE)-mRNAs, 1116 DE-lncRNAs, 54 DE-circRNAs, and 174 DE-miRNAs when comparing myoblasts and myotubes. A GO analysis highlighted the enrichment of DE molecules in the extracellular region, protein binding, and exocyst. A KEGG analysis pinpointed pathways related to ferroptosis, PPAR signaling, nitrogen metabolism, cell cycle, cardiac muscle contraction, glycerolipid metabolism, and actin cytoskeleton. A total of 51 trans-acting lncRNAs, including ENSAPLT00020002101 and ENSAPLT00020012069, were predicted to participate in regulating myoblast proliferation and differentiation. Based on the ceRNAs, we constructed lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA networks involving five miRNAs (miR-129-5p, miR-133a-5p, miR-22-3p, miR-27b-3p, and let-7b-5p) that are relevant to myogenesis. Furthermore, the GO and KEGG analyses of the DE-mRNAs within the ceRNA network underscored the significant enrichment of the glycerolipid metabolism pathway. We identified five different DE-mRNAs, specifically ENSAPLG00020001677, ENSAPLG00020002183, ENSAPLG00020005019, ENSAPLG00020010497, and ENSAPLG00020017682, as potential target genes that are crucial for myogenesis in the context of glycerolipid metabolism. These five mRNAs are integral to ceRNA networks, with miR-107_R-2 and miR-1260 emerging as key regulators. In summary, this study provides a valuable resource elucidating the intricate interplay of mRNA-lncRNA-circRNA-miRNA in duck myogenesis, shedding light on the molecular mechanisms that govern this critical biological process.

Key pathways and genes that are altered during treatment with hyperbaric oxygen in patients with sepsis due to necrotizing soft tissue infection (HBOmic study)

BackgroundFor decades, the basic treatment strategies of necrotizing soft tissue infections (NSTI) have remained unchanged, primarily relying on aggressive surgical removal of infected tissue, broad-spectrum antibiotics, and supportive intensive care. One treatment strategy that has been proposed as an adjunctive measure to improve patient outcomes is hyperbaric oxygen (HBO2) treatment. HBO2 treatment has been linked to several immune modulatory effects; however, investigating these effects is complicated due to the disease's acute life-threatening nature, metabolic and cell homeostasis dependent variability in treatment effects, and heterogeneity with respect to both patient characteristics and involved pathogens. To embrace this complexity, we aimed to explore the underlying biological mechanisms of HBO2 treatment in patients with NSTI on the gene expression level.MethodsWe conducted an observational cohort study on prospective collected data, including 85 patients admitted to the intensive care unit (ICU) for NSTI. All patients were treated with one or two HBO2 treatments and had one blood sample taken before and after the intervention. Total RNAs from blood samples were extracted and mRNA purified with rRNA depletion, followed by whole-transcriptome RNA sequencing with a targeted sequencing depth of 20 million reads. A model for differentially expressed genes (DEGs) was fitted, and the functional aspects of the obtained set of genes was predicted with GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of genes and Genomes) enrichment analyses. All analyses were corrected for multiple testing with FDR.ResultsAfter sequential steps of quality control, a final of 160 biological replicates were included in the present study. We found 394 protein coding genes that were significantly DEGs between the two conditions with FDR < 0.01, of which 205 were upregulated and 189 were downregulated. The enrichment analysis of these DEGs revealed 20 GO terms in biological processes and 12 KEGG pathways that were significantly overrepresented in the upregulated DEGs, of which the term; “adaptive immune response” (GO:0002250) (FDR = 9.88E-13) and “T cell receptor signaling pathway” (hsa04660) (FDR = 1.20E-07) were the most significant. Among the downregulated DEGs two biological processes were significantly enriched, of which the GO term “apoptotic process” (GO:0006915) was the most significant (FDR = 0.001), followed by “Positive regulation of T helper 1 cell cytokine production” (GO:2000556), and “NF-kappa B signaling pathway” (hsa04064) was the only KEGG pathway that was significantly overrepresented (FDR = 0.001).ConclusionsWhen one or two sessions of HBO2 treatment were administered to patients with a dysregulated immune response and systemic inflammation due to NSTI, the important genes that were regulated during the intervention were involved in activation of T helper cells and downregulation of the disease-induced highly inflammatory pathway NF-κB, which was associated with a decrease in the mRNA level of pro-inflammatory factors.Trial registration: Biological material was collected during the INFECT study, registered at ClinicalTrials.gov (NCT01790698).

Plant resistance to tomato yellow leaf curl virus is enhanced by Bacillus amyloliquefaciens Ba13 through modulation of RNA interference.

Tomato yellow leaf curl virus (TYLCV), which is a typical member of the genus Begomovirus, causes severe crop yield losses worldwide. RNA interference (RNAi) is an important antiviral defense mechanism in plants, but whether plant beneficial microbes used as biocontrol agents would modulate RNAi in defense against TYLCV remains unclear. Here, we employed whole-transcriptome, bisulfite, and small RNA sequencing to decipher the possible role of Bacillus amyloliquefaciens Ba13 as a bacterial biocontrol agent against TYLCV in RNAi modulation. Potted tomato plants were exposed to whiteflies for natural viral infection 14 days after bacterial inoculation. Compared with non-inoculated controls, the abundance of TYLCV gene in the leaves of inoculated plants decreased by 70.1% at 28 days post-infection, which mirrored the pattern observed for plant disease index. The expression of the ARGONAUTE family genes (e.g., AGO3, AGO4, AGO5, and AGO7) involved in antiviral defense markedly increased by 2.44-6.73-fold following bacterial inoculation. The methylation level at CpG site 228 (in the open reading frame region of the RNA interference suppressing gene AV2) and site 461 (in the open reading frame regions of AV1 and AV2) was 183.1 and 63.0% higher in inoculated plants than in non-inoculated controls, respectively. The abundances of 10 small interfering RNAs matched to the TYLCV genome were all reduced in inoculated plants, accompanied by enhancement of photosystem and auxin response pathways. The results indicate that the application of Ba. amyloliquefaciens Ba13 enhances plant resistance to TYLCV through RNAi modulation by upregulating RNAi-related gene expression and enhancing viral genome methylation.

Mining host candidate regulators of schistosomiasis-induced liver fibrosis in response to artesunate therapy through transcriptomics approach.

Artesunate (ART) has been reported to have an antifibrotic effect in various organs. The underlying mechanism has not been systematically elucidated. We aimed to clarify the effect of ART on liver fibrosis induced by Schistosoma japonicum (S. japonicum) in an experimentally infected rodent model and the potential underlying mechanisms. The effect of ART on hepatic stellate cells (HSCs) was assessed using CCK-8 and Annexin V-FITC/PI staining assays. The experimental model of liver fibrosis was established in the Mongolian gerbil model infected with S. japonicum cercariae and then treated with 20 mg/kg or 40 mg/kg ART. The hydroxyproline (Hyp) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities in liver tissue were measured and histopathological changes of liver tissues were observed. Whole-transcriptome RNA sequencing (RNA-seq) of the liver tissues was performed. Differentially expressed genes (DEGs) were identified using bioinformatic analysis and verified by quantitative PCR (qPCR) and western blot assay. ART significantly inhibited the proliferation and induce the apoptosis of HSCs in a dose-dependent manner. In vivo, Hyp content decreased significantly in the ART-H group compared to the model (MOD) group and GPX activity was significantly higher in the ART-H group than in the MOD group. Besides, ART treatment significantly reduced collagen production (p <0.05). A total of 158 DEGs and 44 differentially expressed miRNAs related to ART-induced anti-schistosomiasis liver fibrosis were identified. The qPCR and western blot results of selected DEGs were consistent with the sequencing results. These DEGs were implicated in key pathways such as immune and inflammatory response, integrin-mediated signaling and toll-like receptor signaling pathways. ART is effective against liver fibrosis using Mongolian gerbil model induced by S. japonicum infection. We identified host candidate regulators of schistosomiasis-induced liver fibrosis in response to ART through transcriptomics approach.

Alterations in Placental Inflammation-Related Gene Expression Partially Mediate the Effects of Prenatal Alcohol Consumption on Maternal Iron Homeostasis.

Prenatal alcohol exposure (PAE) is associated with alterations in maternal and infant iron homeostasis that are consistent with changes seen in the setting of inflammation. We hypothesized that PAE leads to alterations in the placental expression of genes related to iron metabolism and inflammation that play functional roles in the teratogenic effects of alcohol on iron homeostasis. A total of 126 heavy-drinking women (≥1 oz (30 mL) absolute alcohol/day (~1.67 standard drinks/day) or women reporting binge drinking (≥2 drinks/occasion)) and 80 control women (<0.5 oz AA per day, no binging) in Cape Town, South Africa were interviewed prenatally regarding demographics, and alcohol, smoking, and drug use around conception and during pregnancy. Prenatal/maternal and infant hemoglobin and ferritin were measured. Whole-transcriptome RNA sequencing analysis was performed on flash-frozen transplacental tissue samples. Gene sets related to iron metabolism (n = 398) and inflammation (n = 467) were constructed by searching the Molecular Signatures Database for related ontology terms. Principal component analysis (PCA) yielded 59 factors for each theme. In multivariable regression models, PAE was related to 2 iron metabolism PCA factors (PCs) and 5 inflammation PCs, among which 2 iron metabolism and 4 inflammation factors were related to at least 1 key maternal or infant iron outcome. In causal inference analyses based on marginal structural models and the product method, the alterations in the expression profile of genes with functions in immune cell regulation, cytokine activity, angiogenesis, hematopoiesis, and ubiquitous cell processes appeared to partially mediate the relation of prenatal drinking frequency (days/week) around conception to a lower maternal hemoglobin-to-log(ferritin) ratio (proportion mediation = 51.35%). These findings suggest that placental inflammation may be partly responsible for the differences in alcohol-related iron homeostasis patterns between pregnant and non-pregnant adults.

Different Genetic Signatures of Small-Cell Lung Cancer Characterize Anti-GABAB R and Anti-Hu Paraneoplastic Neurological Syndromes.

Small-cell lung cancer (SCLC) is the malignancy most frequently associated with paraneoplastic neurological syndromes (PNS) and can trigger different antibody responses against intracellular (Hu) or neuronal surface (GABAB R) antigens. Our aim was to clarify whether the genomic and transcriptomic features of SCLC are different in patients with anti-GABAB R or anti-Hu PNS compared with SCLC without PNS. A total of 76 SCLC tumor samples were collected: 34 anti-Hu, 14 anti-GABAB R, and 28 SCLC without PNS. The study consisted of 4 steps: (1) pathological confirmation; (2) next generation sequencing using a panel of 98 genes, including those encoding the autoantibodies targets ELAVL1-4, GABBR1-2, and KCTD16; (3) genome-wide copy number variation (CNV); and (4) whole-transcriptome RNA sequencing. CNV analysis revealed that patients with anti-GABAB R PNS commonly have a gain in chromosome 5q, which contains KCTD16, whereas anti-Hu and control patients often harbor a loss. No significantly different number of mutations regarding any onconeural genes was observed. Conversely, the transcriptomic profile of SCLC was different, and the differentially expressed genes allowed effective clustering of the samples into 3 groups, reflecting the antibody-based classification, with an overexpression of KCTD16 specific to anti-GABAB R PNS. Pathway analysis revealed that tumors of patients with anti-GABAB R encephalitis were enriched in B-cell signatures, as opposed to those of patients with anti-Hu, in which T-cell- and interferon-γ-related signatures were overexpressed. SCLC genetic and transcriptomic features differentiate anti-GABAB R, anti-Hu, and non-PNS tumors. The role of KCTD16 appears to be pivotal in the tumor immune tolerance breakdown of anti-GABAB R PNS. ANN NEUROL 2023;94:1102-1115.

Molecular mechanisms that regulate the heat stress response in sea urchins (Strongylocentrotus intermedius) by comparative heat tolerance performance and whole-transcriptome RNA sequencing

In the context of climate change and extreme high temperature, the commercially important sea urchin Strongylocentrotus intermedius suffers high mortality during summer in Northern China. How sea urchins respond to high temperatures is of great concern to academia and industry. How to understand the heat tolerance of sea urchin from the whole transcriptome level. In this study, the heat-resistant S. intermedius bred by our team and its control group were used as the research objects, then we applied whole-transcriptome RNA sequencing to detect differentially expressed mRNAs, microRNAs, long noncoding RNAs that respond to heat stress in the heat-resistant and control S. intermedius. A competitive endogenous RNA (ceRNA) regulatory network was constructed with predicted pairs of differentially expressed mRNAs and noncoding RNAs and revealed the molecular regulatory mechanisms in S. intermedius responding to heat stress. A functional analysis suggested that the ceRNAs were involved in basal metabolism, calcium ion transport, endoplasmic reticulum stress, and apoptosis. This is the whole-transcriptomic analysis of S. intermedius under heat stress to propose ceRNA networks that will provide a basis for studying the potential functions of long noncoding RNAs and miRNAs in the heat stress response in S. intermedius and provide a theoretical basis for the study of the molecular mechanism of sea urchins in response to environmental changes.

Chidamide and Oxaliplatin Synergistically Inhibit Colorectal Cancer Growth by Regulating the RPS27A-MDM2-P53 Axis.

The present study explored the anti-tumor effects of chidamide plus oxaliplatin on colorectal cancer (CRC) and examined its underlying mechanism. First, the Combination Index (CI) of chidamide and oxaliplatin was evaluated via CCK-8 assay. Second, the effects of chidamide and oxaliplatin monotherapy and the combined treatment on cell proliferation, invasion, migration, and apoptosis were detected. Third, whole-transcriptome RNA sequencing (RNA-seq) was performed to seek the potential targeted gene by which chidamide plus oxaliplatin exerted anti-tumor effects. Fourth, the validation of the targeted gene and the signal pathway it regulated were performed. Finally, the anti-tumor effect of chidamide plus oxaliplatin on mice xenograft was examined. Chidamide and oxaliplatin acted synergistically to inhibit CRC growth in vitro and in vivo (CI<1). Besides, compared with oxaliplatin monotherapy, chidamide could significantly enhance oxaliplatin-induced inhibition in cell proliferation, invasion, and migration, and promotion in HCT-116 and RKO cell apoptosis (P<0.05). The RNA-seq displayed that, compared to oxaliplatin monotherapy, RPS27A mRNA was evidently decreased in HCT-116 cells treated with chidamide plus oxaliplatin (P<0.001). Then, we found RPS27A was highly expressed in CRC tissues and CRC cell lines (P<0.001). Silence of RPS27A attenuated proliferation and induced apoptosis in HCT-116 and RKO cells via downregulation of MDM2 expression and upregulation of P53. Next, RPS27A overexpression could partially reverse chidamide plus oxaliplatin induced growth inhibition and apoptosis in HCT-116 and RKO cells (P<0.01). RPS27A overexpression could promote the upregulation of MDM2 and downregulation of P53 after the combined treatment of chidamide with oxaliplatin. Chidamide and oxaliplatin acted synergistically to suppress CRC growth by the inhibition of the RPS27A-MDM2-p53 axis.

Integrated clinical, pathologic, and genetic data in a racially diverse hereditary breast cancer cohort.

10553 Background: Hereditary breast cancers (HBC) represent 5-10% of breast cancer, predominantly due to pathogenic/likely pathogenic (P/LP) variants in the BRCA1 and BRCA2 genes and other high (e.g., PALB2) and moderate (e.g., ATM and CHEK2) penetrance genes. There remains a paucity of data evaluating the role of germline-somatic interactions and possible confounders on breast cancer outcomes. Furthermore, current knowledge about HBC is predominantly defined in the context of European ancestry. We sought to characterize differences in clinicopathological features and somatic mutations across P/LP variants in a racially diverse HBC cohort. Methods: Among females with breast cancer and a confirmed P/LP variant in BRCA1, BRCA2, PALB2, CHEK2, and/or ATM currently enrolled through existing Vanderbilt-based studies (ICARE: Inherited Cancer Registry and BEST: Black Women: Etiology and Survival of Triple Negative Breast Cancers (TNBC)), we abstracted data from medical records and performed tumor sequencing using the Tempus xT assay (a 596-gene DNA next-generation sequencing panel and whole-transcriptome RNA sequencing). Interim differences in clinicopathological features were analyzed using Pearson’s chi-square test and the frequency of somatic mutations reported by gene. Results: To date, we have data from 91 females with breast cancer (77% White and 23% Black) and a P/LP variant (36 BRCA1, 27 BRCA2, 13 PALB2, 11 CHEK2 and 4 ATM). The median age at diagnosis was 44 (38-50), 85% had localized breast cancer (Stage I-II), and 69% had Grade 3 tumors. Significantly more Black participants were BRCA1 carriers (p = 0.008), with no significant racial differences observed in other genes. There were significant differences in IHC subtype by gene (p &lt; 0.001); with higher frequency of TNBC in BRCA1 carriers (64%) whereas BRCA2, PALB2, CHEK2, and ATM carriers were more likely to have HR+, HER2- breast cancer (67%, 54%, 73%, 75%, respectively). Of the 85% who received chemotherapy, significantly higher rates were observed in BRCA1 carriers (p = 0.02). Most participants opted to proceed with a risk reducing mastectomy (69%) with no difference across genes (p = 0.91). Higher rates of somatic TP53 mutations were observed among BRCA1, BRCA2, PALB2 carriers (89%, 56%, 46%) but not as frequent among ATM (25%) and CHEK2 (9%) carriers. Conclusions: These preliminary results show that among a diverse cohort of females with HBC, BRCA1 mutations were overrepresented among Black females and those with TNBC. BRCA1, BRCA2, and PALB2 carriers had a higher prevalence of somatic TP53 mutations, which are typically associated with more aggressive tumor biology and worse survival outcomes. Current efforts underway include full genomic and transcriptomic analysis, through which we will be able to improve our understanding of breast cancer clinicopathological characteristics and survival outcomes among a diverse cohort of females with HBC.

Complete variable domain sequences of monoclonal antibody light chains identified from untargeted RNA sequencing data.

Monoclonal antibody light chain proteins secreted by clonal plasma cells cause tissue damage due to amyloid deposition and other mechanisms. The unique protein sequence associated with each case contributes to the diversity of clinical features observed in patients. Extensive work has characterized many light chains associated with multiple myeloma, light chain amyloidosis and other disorders, which we have collected in the publicly accessible database, AL-Base. However, light chain sequence diversity makes it difficult to determine the contribution of specific amino acid changes to pathology. Sequences of light chains associated with multiple myeloma provide a useful comparison to study mechanisms of light chain aggregation, but relatively few monoclonal sequences have been determined. Therefore, we sought to identify complete light chain sequences from existing high throughput sequencing data. We developed a computational approach using the MiXCR suite of tools to extract complete rearranged IGVL-IGJL sequences from untargeted RNA sequencing data. This method was applied to whole-transcriptome RNA sequencing data from 766 newly diagnosed patients in the Multiple Myeloma Research Foundation CoMMpass study. Monoclonal IGVL-IGJL sequences were defined as those where >50% of assigned IGK or IGL reads from each sample mapped to a unique sequence. Clonal light chain sequences were identified in 705/766 samples from the CoMMpass study. Of these, 685 sequences covered the complete IGVL-IGJL region. The identity of the assigned sequences is consistent with their associated clinical data and with partial sequences previously determined from the same cohort of samples. Sequences have been deposited in AL-Base. Our method allows routine identification of clonal antibody sequences from RNA sequencing data collected for gene expression studies. The sequences identified represent, to our knowledge, the largest collection of multiple myeloma-associated light chains reported to date. This work substantially increases the number of monoclonal light chains known to be associated with non-amyloid plasma cell disorders and will facilitate studies of light chain pathology.