Integrated Analysis Of Gene Expression Research Articles

Integrative Analysis of Small RNA and Gene Expression Uncovers a Novel Long-Noncoding MicroRNA Targeting a UDP-Transferase in Camellia chekiangoleosa.

Biosynthesis of specific secondary metabolites in plants involves fine regulation of gene expression. Camellia chekiangoleosa has important economic value: the seeds contain high-quality unsaturated fatty acids and the pericarp is rich in tea saponins. As an important posttranscriptional regulator, the role of microRNAs (miRNAs) in controlling secondary metabolism in C. chekiangoleosa is not fully studied. Here, we investigated the role of miRNAs and their targets in the secondary metabolic regulatory network by comprehensively analyzing small RNAs, transcriptomes, and degradomes from different tissues. We identified 168 known miRNAs and 74 novel miRNAs in the C. chekiangoleosa genome and revealed 15 tandem clusters containing 35 miRNAs. By establishing a gene regulatory network containing miRNAs, target genes, and transcription factors, we unravelled the multiplicity of miRNA tissue-specific regulation of gene expression, which may be tightly linked to the synthesis of secondary metabolites. Furthermore, we characterized a novel long-noncoding miRNA gene (cch-miR3633) that targeted a UDP-transferase gene (CchUGT94E5). We showed that, ectopic expression of CchUGT94E5 caused outgrowth of shoot branching and changes in cytokinin contents in Arabidopsis, indicating a potential role of regulating secondary metabolism. This work provides valuable information for the study of miRNA regulation of secondary metabolism.

OS03-10 Early biomarkers of cancer susceptibility in benzene-exposed workers: integrated analysis of gene expression, microRNA alterations, and DNA methylation datasets

OS03-10 Early biomarkers of cancer susceptibility in benzene-exposed workers: integrated analysis of gene expression, microRNA alterations, and DNA methylation datasets

Dysfunctional tumor-infiltrating Vδ1 + T lymphocytes in microsatellite-stable colorectal cancer

Although γδ T cells are known to participate in immune dysregulation in solid tumors, their relevance to human microsatellite-stable (MSS) colorectal cancer (CRC) is still undefined. Here, using integrated gene expression analysis and T cell receptor sequencing, we characterized γδ T cells in MSS CRC, with a focus on Vδ1 + T cells. We identified Vδ1+ T cells with shared motifs in the third complementarity-determining region of the δ-chain, reflective of antigen recognition. Changes in gene and protein expression levels suggested a dysfunctional effector state of Vδ1+ T cells in MSS CRC, distinct from Vδ1+ T cells in microsatellite-instable (MSI). Interaction analysis highlighted an immunosuppressive role of fibroblasts in the dysregulation of Vδ1+ T cells in MSS CRC via the TIGIT-NECTIN2 axis. Blocking this pathway with a TIGIT antibody partially restored cytotoxicity of the dysfunctional Vδ1 phenotype. These results define an operative pathway in γδ T cells in MSS CRC.

Identification of potential diagnostic biomarkers for hypertension via integrated analysis of gene expression and DNA methylation

Objective Hypertension refers to the elevated blood pressure (BP) in arteries, with a BP reading of 140/90 mm Hg or higher in adults. Over 40% of >25-year-old population have suffered from hypertension. Thus, this study aimed to find novel diagnostic biomarkers for hypertension. Methods All hypertension-related mRNA and methylation datasets were downloaded from the GEO database. Liner model method was used to identify differentially expressed genes (DEGs) between hypertension and control groups. Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes enrichment analysis was employed to obtain functional information. CpG sites and the corresponding genes associated with hypertension were screened using epigenome-wide association study (EWAS) analysis. Results There were 37 DEGs between the hypertension group and control group, which were significantly enriched in 84 Biological Process terms, 31 Cellular Component terms, 18 Molecular Function terms and 9 signalling pathways. EWAS results indicated that 1072 CpG sites were associated with hypertension occurrence, corresponding to 1029 genes. After cross-analysis, complement factor D (CFD) and OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2) with methylation modification were identified as diagnostic markers for hypertension. Conclusion In conclusion, CFD and OTUB2 were potential biomarkers of hypertension occurrence. Our results will provide more information for hypertension diagnosis and would be more reliable combined with multiple biomarkers.

Optimizing small-diameter vascular grafts: Harnessing the power of recombinant human type III collagen (rhCOLIII) for enhanced antiplatelet and endothelialization performance

Small-diameter vascular grafts hold great promise as potential substitutes for coronary and peripheral arteries. However, their clinical availability is hindered by thrombogenicity and intimal hyperplasia. Herein, bilayer vascular grafts with enhanced antithrombogenicity and endothelialization were developed by two-step electrospinning technology. First, the well-designed recombinant human type III collagen (rhCOLIII), featuring numerous triplets that promote cell adhesion while excluding sequences prone to platelet adhesion and activation, was composited with poly(L-lactide-co-ε-caprolactone) (PLCL) to create a bioactive inner layer. Afterward, a pure PLCL outer layer with exceptional elasticity was constructed to provide mechanical support. In vitro antiplatelet and cytocompatibility experiments revealed that the graft containing 60% rhCOLIII exhibited almost no platelet adhesion, while the number of adhered endothelial cells increased approximately 20 times compared to pure PLCL. Furthermore, Beagle dog femoral artery implantation models demonstrated that the rhCOLIII-modified grafts underwent endothelialization within three months, contributing to the patency of grafts. The potential mechanism for enhanced endothelialization by rhCOLIII was investigated through transcriptome sequencing and verified by an integrated analysis of gene and protein expression. The cell-affinity units, Gly-Glu-Arg (GER) and Gly-Glu-Lys (GEK) triplets, present in rhCOLIII contribute to initial adhesion of endothelial cells. Following adhesion, they may upregulate the expression of integrins αVβ8 on the cell surface, thereby further enhancing cell adhesion. Subsequently, the FAK/PI3K/AKT pathway within endothelial cells may also be activated to promote cell proliferation.

Multi-platform sequencing explores the transcriptomics of terpenoid biosynthesis in Melaleuca alternifolia

Leaves of Melaleuca alternifolia are abundant in essential oils comprising terpenoids and volatile organic compounds, which play an important role in plant abiotic stress response. However, the molecular mechanisms underlying terpenoid biosynthesis in M. alternifolia remain poorly understood. To fill this knowledge gap, we profiled terpenoid content and composition across four distinct growth phases using gas chromatography-mass spectrometry (GC/MS). We also performed an integrated analysis of gene expression in leaf tissues through full-length transcriptome sequencing combined with high throughput RNA sequencing. We identified 46,313 transcripts, 99 % (45,848 transcripts) of which were annotated using existing databases. Our study also revealed 2,792 transcription factors (TFs) and 1,812 alternative splicing (AS) events. Through weighted gene co-expression network analysis (WGCNA), we clustered these identified genes into 11 co-expressed modules. Notably, two modules—designated as blue and brown—were significantly correlated with essential oil content. Homologous annotation with the Arabidopsis database revealed that genes within the blue module are largely involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, suggesting its pivotal role in essential oil synthesis in M. alternifolia. Furthermore, elevated expression levels of VAMP72 and DGK1 in the brown module indicate their potential function as negative regulators of essential oil synthesis. Our findings unveil key differentially expressed genes (DEGs) and pathways that can be targeted to enhance essential oil production in M. alternifolia. The identified TFs may serve as potential candidates for genetic modification through genome editing technologies. This research provides valuable insights into the processes involved in essential oil biosynthesis and highlights key genes for functional characterization in M. alternifolia.

Integrative gene expression analysis and animal model reveal immune- and autophagy-related biomarkers in osteomyelitis.

Osteomyelitis (OM) is recognized as a significant challenge in orthopedics due to its complex immune and inflammatory responses. The prognosis heavily depends on timely diagnosis, accurate classification, and assessment of severity. Thus, the identification of diagnostic and classification-related genes from an immunological standpoint is crucial for the early detection and tailored treatment of OM. Transcriptomic data for OM was sourced from the Gene Expression Omnibus(GEO) database, leading to the identification of autophagy- and immune-related differentially expressed genes (AIR-DEGs) through differential expression analysis. Diagnostic and classification models were subsequently developed. The CIBERSORT algorithm was utilized to examine immune cell infiltration in OM, and the relationship between OM clusters and various immune cells was explored. Key AIR-DEGs were further validated through the creation of OM animal models. Analysis of the transcriptomic data revealed three AIR-DEGs that played a significant role in immune responses and pathways. Nomogram and receiver operating characteristic curve analyses were performed, demonstrating excellent diagnostic capability for differentiating between OM patients and healthy individuals, with an area under the curveof 0.814. An unsupervised clustering analysis discerned two unique patterns of autophagy- and immune-related genes,as well as gene patterns. Further exploration into immune infiltration exhibited notable variances across different subtypes, especially between OM cluster 1 and gene cluster A, highlighting their potential role in mitigating inflammatory responses by regulating immune activities. Moreover, the mRNA and protein expression levels of three AIR-DEGs in the animal model were aligned with those in the training and validation data sets. From an immunological perspective, a diagnostic model was successfully developed, and two distinct clustering patterns were identified. These contributions offer a significant resource for the early detection and personalized immunotherapy of patients with OM.

Deconstructing Intratumoral Heterogeneity through Multiomic and Multiscale Analysis of Serial Sections.

Tumors may contain billions of cells, including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that are consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.

Integrative Genetic Variation, DNA Methylation, and Gene Expression Analysis of Escitalopram and Aripiprazole Treatment Outcomes in Depression: A CAN-BIND-1 Study.

Little is known about the interplay between genetics and epigenetics on antidepressant treatment (1) response and remission, (2) side effects, and (3) serum levels. This study explored the relationship among single nucleotide polymorphisms (SNPs), DNA methylation (DNAm), and mRNA levels of four pharmacokinetic genes, CYP2C19, CYP2D6, CYP3A4, and ABCB1, and its effect on these outcomes. The Canadian Biomarker Integration Network for Depression-1 dataset consisted of 177 individuals with major depressive disorder treated for 8 weeks with escitalopram (ESC) followed by 8 weeks with ESC monotherapy or augmentation with aripiprazole. DNAm quantitative trait loci (mQTL), identified by SNP-CpG associations between 20 SNPs and 60 CpG sites in whole blood, were tested for associations with our outcomes, followed by causal inference tests (CITs) to identify methylation-mediated genetic effects. Eleven cis-SNP-CpG pairs (q<0.05) constituting four unique SNPs were identified. Although no significant associations were observed between mQTLs and response/remission, CYP2C19 rs4244285 was associated with treatment-related weight gain (q=0.027) and serum concentrations of ESCadj (q<0.001). Between weeks 2-4, 6.7% and 14.9% of those with *1/*1 (normal metabolizers) and *1/*2 (intermediate metabolizers) genotypes, respectively, reported ≥2 lbs of weight gain. In contrast, the *2/*2 genotype (poor metabolizers) did not report weight gain during this period and demonstrated the highest ESCadj concentrations. CITs did not indicate that these effects were epigenetically mediated. These results elucidate functional mechanisms underlying the established associations between CYP2C19 rs4244285 and ESC pharmacokinetics. This mQTL SNP as a marker for antidepressant-related weight gain needs to be further explored.

HyperGCN: an effective deep representation learning framework for the integrative analysis of spatial transcriptomics data

BackgroundAdvances of spatial transcriptomics technologies enabled simultaneously profiling gene expression and spatial locations of cells from the same tissue. Computational tools and approaches for integration of transcriptomics data and spatial context information are urgently needed to comprehensively explore the underlying structure patterns. In this manuscript, we propose HyperGCN for the integrative analysis of gene expression and spatial information profiled from the same tissue. HyperGCN enables data visualization and clustering, and facilitates downstream analysis, including domain segmentation, the characterization of marker genes for the specific domain structure and GO enrichment analysis.ResultsExtensive experiments are implemented on four real datasets from different tissues (including human dorsolateral prefrontal cortex, human positive breast tumors, mouse brain, mouse olfactory bulb tissue and Zabrafish melanoma) and technologies (including 10X visium, osmFISH, seqFISH+, 10X Xenium and Stereo-seq) with different spatial resolutions. The results show that HyperGCN achieves superior clustering performance and produces good domain segmentation effects while identifies biologically meaningful spatial expression patterns. This study provides a flexible framework to analyze spatial transcriptomics data with high geometric complexity.ConclusionsHyperGCN is an unsupervised method based on hypergraph induced graph convolutional network, where it assumes that there existed disjoint tissues with high geometric complexity, and models the semantic relationship of cells through hypergraph, which better tackles the high-order interactions of cells and levels of noise in spatial transcriptomics data.

Integrative Metabolomics, Enzymatic Activity, and Gene Expression Analysis Provide Insights into the Metabolic Profile Differences between the Slow-Twitch Muscle and Fast-Twitch Muscle of Pseudocaranx dentex.

The skeletal muscles of teleost fish encompass heterogeneous muscle types, termed slow-twitch muscle (SM) and fast-twitch muscle (FM), characterized by distinct morphological, anatomical, histological, biochemical, and physiological attributes, driving different swimming behaviors. Despite the central role of metabolism in regulating skeletal muscle types and functions, comprehensive metabolomics investigations focusing on the metabolic differences between these muscle types are lacking. To reveal the differences in metabolic characteristics between the SM and FM of teleost, we conducted an untargeted metabolomics analysis using Pseudocaranx dentex as a representative model and identified 411 differential metabolites (DFMs), of which 345 exhibited higher contents in SM and 66 in FM. KEGG enrichment analysis showed that these DFMs were enriched in the metabolic processes of lipids, amino acids, carbohydrates, purines, and vitamins, suggesting that there were significant differences between the SM and FM in multiple metabolic pathways, especially in the metabolism of energy substances. Furthermore, an integrative analysis of metabolite contents, enzymatic activity assays, and gene expression levels involved in ATP-PCr phosphate, anaerobic glycolysis, and aerobic oxidative energy systems was performed to explore the potential regulatory mechanisms of energy metabolism differences. The results unveiled a set of differential metabolites, enzymes, and genes between the SM and FM, providing compelling molecular evidence of the FM achieving a higher anaerobic energy supply capacity through the ATP-PCr phosphate and glycolysis energy systems, while the SM obtains greater energy supply capacity via aerobic oxidation. These findings significantly advance our understanding of the metabolic profiles and related regulatory mechanisms of skeletal muscles, thereby expanding the knowledge of metabolic physiology and ecological adaptation in teleost fish.

CircHIRA sponges miR-196b-5p to promote porcine early embryonic development

Circular RNA (circRNA) is abundantly expressed in preimplantation embryos and embryonic stem cells in mice and humans. However, its function and mechanism in early development of mammalian embryos remain unclear. Here, we showed that circHIRA mediated miR-196b-5p to regulate porcine early embryonic development. We verified the circular feature of circHIRA by sanger sequencing, and proved the authenticity of circHIRA by enzyme digestion test. HIRA and circHIRA were expressed in porcine early embryos, and its expression levels significantly increased from 8-cell stage onwards and reached the maximum at the blastocyst stage. Functional studies revealed that circHIRA knockdown not only significantly reduced the developmental efficiency of embryos from 8-cell stage to blastocyst stage, but also impaired the blastocyst quality. Mechanistically, integrated analysis of miRNA prediction and gene expression showed that circHIRA knockdown significantly increased the expression of miR-196b-5p in porcine early embryos. Furthermore, miR-196b-5p inhibitor injection could rescue the early development of circHIRA knockdown embryos. Taken together, the findings reveal that circHIRA regulates porcine early embryonic development via inhibiting the expression of miR-196b-5p.

Abstract 3067: Chronic Knockout Of The ETS Transcription Factor ERG Promotes Loss Of Endothelial Cell Identity And Endothelial Cell Dysfunction In An Aortic Endothelial Cell Model Relevant For Atherosclerosis

Background: The ETS transcription factor ERG is a key endothelial cell (EC) transcription factor. ERG represses vascular inflammation and is downregulated in chronic inflammatory diseases (e.g. liver fibrogenesis, atherosclerosis). Previously, transient ERG knock-down has been associated with endothelial-mesenchymal transition (EndMT) and EC dysfunction; however, the mechanisms underlying EndMT and the EC dysfunction associated with chronic ERG loss have not been elucidated in an atherosclerosis-relevant model. We hypothesize that chronic ERG knockout (KO) in telomerase-immortalized human aortic ECs (teloHAECs) induces EndMT and contributes to EC dysfunction underlying vascular dysregulation in atherosclerosis. Methods: TeloHAECs with CRIPSR/Cas9-mediated deletion of ERG were used. Chromatin accessibility and gene expression in ERG KO and wildtype (WT) teloHAECs were assessed using ATAC-seq (n=2) and RNA-seq (n=5-6). TOBIAS and diffTF were used to identify enriched motifs by footprinting (P&lt;0.05) and to profile TF activity (FDR&lt;0.05), respectively. ERG-mediated EC dysfunction was investigated using immunofluorescence (IF; n=3), western blot (WB; n=3), and a timelapse migration assay (n=3). Results: There were ~ 21,000 differentially accessible chromatin peaks between ERG KO and WT ECs. Integrated analysis of chromatin accessibility and gene expression demonstrated increased activity of TGFβ-SMAD and IL-1β signaling constituents and classical EndMT transcription factors. Loss of ERG downregulated EC genes (e.g., CLDN5 , TIE1 ) and protein expression (e.g., CDH5, vWF) and upregulated mesenchymal genes (e.g., ACTA2 , FSP1 ) and protein distribution (e.g., FN1, COL1A1). Investigation of EC-dysfunction phenotypes revealed increased migration and proliferation in ERG KO teloHAEC - with the percentage of Ki67 positive ECs increasing from 14% to 49% (P&lt;0.05, n=3). Conclusion: ERG KO in cultured aortic ECs markedly impacts chromatin accessibility and gene expression and induces EC identity loss, migration, and proliferation, providing mechanistic insight into the role of ERG in vascular dysfunction in atherosclerosis.

Hypomethylation of ATP1A1 Is Associated with Poor Prognosis and Cancer Progression in Triple-Negative Breast Cancer.

Dysregulated DNA methylation in cancer is critical in the transcription machinery associated with cancer progression. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, but no treatment targeting TNBC biomarkers has yet been developed. To identify specific DNA methylation patterns in TNBC, methyl-binding domain protein 2 (MBD) sequencing data were compared in TNBC and the three other major breast cancer subtypes. Integrated analysis of DNA methylation and gene expression identified a gene set showing a correlation between DNA methylation and gene expression. ATPase Na+/K+-transporting subunit alpha 1 (ATP1A1) was found to be specifically hypomethylated in the coding sequence (CDS) region and to show increased expression in TNBC. The Cancer Genome Atlas (TCGA) database also showed that hypomethylation and high expression of ATP1A1 were strongly associated with poor survival in patients with TNBC. Furthermore, ATP1A1 knockdown significantly reduced the viability and tumor-sphere formation of TNBC cells. These results suggest that the hypomethylation and overexpression of ATP1A1 could be a prognostic marker in TNBC and that the manipulation of ATP1A1 expression could be a therapeutic target in this disease.

The genome regulatory landscape of Atlantic salmon liver through smoltification.

The anadromous Atlantic salmon undergo a preparatory physiological transformation before seawater entry, referred to as smoltification. Key molecular developmental processes involved in this life stage transition, such as remodeling of gill functions, are known to be synchronized and modulated by environmental cues like photoperiod. However, little is known about the photoperiod influence and genome regulatory processes driving other canonical aspects of smoltification such as the large-scale changes in lipid metabolism and energy homeostasis in the developing smolt liver. Here we generate transcriptome, DNA methylation, and chromatin accessibility data from salmon livers across smoltification under different photoperiod regimes. We find a systematic reduction of expression levels of genes with a metabolic function, such as lipid metabolism, and increased expression of energy related genes such as oxidative phosphorylation, during smolt development in freshwater. However, in contrast to similar studies of the gill, smolt liver gene expression prior to seawater transfer was not impacted by photoperiodic history. Integrated analyses of gene expression, chromatin accessibility, and transcription factor (TF) binding signatures highlight chromatin remodeling and TF dynamics underlying smolt gene regulatory changes. Differential peak accessibility patterns largely matched differential gene expression patterns during smoltification and we infer that ZNF682, KLFs, and NFY TFs are important in driving a liver metabolic shift from synthesis to break down of organic compounds in freshwater. Overall, chromatin accessibility and TFBS occupancy were highly correlated to changes in gene expression. On the other hand, we identified numerous differential methylation patterns across the genome, but associated genes were not functionally enriched or correlated to observed gene expression changes across smolt development. Taken together, this work highlights the relative importance of chromatin remodeling during smoltification and demonstrates that metabolic remodeling occurs as a preadaptation to life at sea that is not to a large extent driven by photoperiod history.

Prioritizing susceptibility genes for the prognosis of male-pattern baldness with transcriptome-wide association study

BackgroundMale-pattern baldness (MPB) is the most common cause of hair loss in men. It can be categorized into three types: type 2 (T2), type 3 (T3), and type 4 (T4), with type 1 (T1) being considered normal. Although various MPB-associated genetic variants have been suggested, a comprehensive study for linking these variants to gene expression regulation has not been performed to the best of our knowledge.ResultsIn this study, we prioritized MPB-related tissue panels using tissue-specific enrichment analysis and utilized single-tissue panels from genotype-tissue expression version 8, as well as cross-tissue panels from context-specific genetics. Through a transcriptome-wide association study and colocalization analysis, we identified 52, 75, and 144 MPB associations for T2, T3, and T4, respectively. To assess the causality of MPB genes, we performed a conditional and joint analysis, which revealed 10, 11, and 54 putative causality genes for T2, T3, and T4, respectively. Finally, we conducted drug repositioning and identified potential drug candidates that are connected to MPB-associated genes.ConclusionsOverall, through an integrative analysis of gene expression and genotype data, we have identified robust MPB susceptibility genes that may help uncover the underlying molecular mechanisms and the novel drug candidates that may alleviate MPB.

Rab4A-directed endosome traffic shapes pro-inflammatory mitochondrial metabolism in T cells via mitophagy, CD98 expression, and kynurenine-sensitive mTOR activation

Activation of the mechanistic target of rapamycin (mTOR) is a key metabolic checkpoint of pro-inflammatory T-cell development that contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), however, the underlying mechanisms remain poorly understood. Here, we identify a functional role for Rab4A-directed endosome traffic in CD98 receptor recycling, mTOR activation, and accumulation of mitochondria that connect metabolic pathways with immune cell lineage development and lupus pathogenesis. Based on integrated analyses of gene expression, receptor traffic, and stable isotope tracing of metabolic pathways, constitutively active Rab4AQ72L exerts cell type-specific control over metabolic networks, dominantly impacting CD98-dependent kynurenine production, mTOR activation, mitochondrial electron transport and flux through the tricarboxylic acid cycle and thus expands CD4+ and CD3+CD4−CD8− double-negative T cells over CD8+ T cells, enhancing B cell activation, plasma cell development, antinuclear and antiphospholipid autoantibody production, and glomerulonephritis in lupus-prone mice. Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism and lineage skewing and attenuate glomerulonephritis. This study identifies Rab4A-directed endosome traffic as a multilevel regulator of T cell lineage specification during lupus pathogenesis.

IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

Vascular endothelial growth factor tyrosine kinase inhibitors (TKIs) have been the standard of care for advanced and metastatic clear cell renal cell carcinoma (ccRCC). However, the therapeutic effect of TKI monotherapy remains unsatisfactory given the high rates of acquired resistance to TKI therapy despite favorable initial tumor response. To define the TKI-resistance mechanism and identify new therapeutic target for TKI-resistant ccRCC, an integrative differential gene expression analysis was performed using acquired resistant cohort and a public dataset. Sunitinib-resistant RCC cell lines were established and used to test their malignant behaviors of TKI resistance through in vitro and in vivo studies. Immunohistochemistry was conducted to compare expression between the tumor and normal kidney and verify expression of pathway-related proteins. Integrated differential gene expression analysis revealed increased interferon-induced transmembrane protein 3 (IFITM3) expression in post-TKI samples. IFITM3 expression was increased in ccRCC compared with the normal kidney. TKI-resistant RCC cells showed high expression of IFITM3 compared with TKI-sensitive cells and displayed aggressive biologic features such as higher proliferative ability, clonogenic survival, migration, and invasion while being treated with sunitinib. These aggressive features were suppressed by the inhibition of IFITM3 expression and promoted by IFITM3 overexpression, and these findings were confirmed in a xenograft model. IFITM3-mediated TKI resistance was associated with the activation of TRAF6 and MAPK/AP-1 pathways. These results demonstrate IFITM3-mediated activation of the TRAF6/MAPK/AP-1 pathways as a mechanism of acquired TKI resistance, and suggest IFITM3 as a new target for TKI-resistant ccRCC.

SPI1 Is the Master Regulator of the Small Cell Variant of Anaplastic Large Cell Lymphoma Controlled By Methylation of SPI1 Gene Promoter Region

Introduction The small cell variant of anaplastic large cell lymphoma (SC-ALCL) is a subtype of anaplastic lymphoma kinase (ALK)-positive ALCL characterized by chemoresistance and poor prognosis, necessitating novel treatment strategies. Recent advances in targeted therapies have resulted in significant responses in various chemoresistant hematological malignancies. Therefore, understanding the underlying oncogenic mechanisms and identifying potential therapeutic targets are critical for overcoming SC-ALCL. Immunohistochemically, SC-ALCL comprises two distinct tumor cell populations: small tumor cells demonstrating negative to weak ALK and CD30 protein expression, and large tumor cells with robust expression of these proteins. We previously reported that small tumor cells display particular resistance to chemotherapy; however, the mechanisms underlying chemoresistance and the molecular characteristics of small tumor cells remain elusive. This study aimed to elucidate the molecular attributes of small tumor cells and pave the way for the development of innovative treatment approaches. Methods We investigated the characteristics of small tumor cells using patient blood samples diagnosed with SC-ALCL leukemic presentation and publicly available RNA-seq data from eight ALK-positive ALCL cell lines. Small and large tumor cells were separated from blood samples based on CD30 expression using CD30 - a phycoerythrin antibody and a magnetic bead separation kit. The presence of the NPM-ALK fusion gene was confirmed in both populations by PCR analysis. Bulk RNA-seq analysis was performed on four samples: small and large tumor cells at the onset and at the first (mainly occupied by small tumor cells) and second (mainly occupied by large tumor cells) relapses. Gene set enrichment analysis was used to identify gene expression patterns in small tumor cells. Signature genes of small tumor cells were identified by filtering with the following thresholds: false discovery rate-adjusted P-value &amp;lt;0.05, and fold-change &amp;gt;2 compared to large tumor cells. The transcription factors regulating the signature genes of small tumor cells were predicted using the g:Profiler software. Result Bulk RNA sequencing analysis of the four blood samples revealed that the SPI1 gene was significantly upregulated in small tumor cells compared to that in large tumor cells. SPI1 has emerged as the highest-scoring transcription factor, regulating the expression of 570 genes that characterize small tumor cells. Approximately 70% of these signature genes overlapped with the SPI1 target genes. Furthermore, gene set enrichment analysis revealed that small tumor cells display increased anti-apoptotic gene activity and decreased cell proliferation gene activity. These observations were validated by integrated gene expression analysis using publicly available RNA-seq data from eight ALK-positive ALCL cell lines (Figure 1). The study found high expression of SPI1 and SPI1 targeted genes in small tumor cells, accompanied by low expression of anti-apoptotic genes in ALK-positive ALCL cell lines, which were more analogous to large tumor cells than small tumor cells. Single-cell RNA-seq analysis validated the high expression of the SPI1 gene in small tumor cells. To explore the mechanism regulating SPI1 gene expression, we examined DNA methylation patterns in the SPI1 promoter region using conventional bisulfite sequencing. Conventional bisulfite sequencing revealed a hypomethylated SPI1 promoter region in small tumor cells and a hypermethylated region in large tumor cells, potentially accounting for the differential SPI1 expression. Conclusion This study highlights SPI1gene as the master regulator determining the unique characteristics of small tumor cells in SC-ALCL. Furthermore, the regulation of SPI1 gene expression appeared to be mediated by DNA methylation patterns within the SPI1 promoter region. These findings pave the way for innovative treatment strategies for SC-ALCL such as SPI1 targeting therapy and methylation-based therapies, offering promising avenues for treating SC-ALCL.

Multi-omic analysis of human kidney tissue identified medulla-specific gene expression patterns

The kidney medulla is a specialized region with important homeostatic functions. It has been implicated in genetic and developmental disorders along with ischemic and drug-induced injuries. Despite its role in kidney function and disease, the medulla's baseline gene expression and epigenomic signatures have not been well described in the adult human kidney. Here we generated and analyzed gene expression (RNA-seq), chromatin accessibility (ATAC-seq), chromatin conformation (Hi-C) and spatial transcriptomic data from the adult human kidney cortex and medulla. Tissue samples were obtained from macroscopically dissected cortex and medulla of tumor-adjacent normal material in nephrectomy specimens from five male patients. We used these carefully annotated specimens to reassign incorrectly labeled samples in the larger public Genotype-Tissue Expression (GTEx) Project, and to extract meaningful medullary gene expression signatures. Using integrated analysis of gene expression, chromatin accessibility and conformation profiles, we found insights into medulla development and function and then validated this by spatial transcriptomics and immunohistochemistry. Thus, our datasets provide a valuable resource for functional annotation of variants from genome-wide association studies and are freely accessible through an epigenome browser portal.