Transcriptome Profiling Of Cells Research Articles

Transcriptome Profiling Analysis of Breast Cancer Cell MCF-7 Treated by Sesamol

Breast cancer is a highly malignant tumor that affects a large number of women worldwide. Sesamol, a natural compound, has been shown to exhibit inhibitory effects on various tumors, including breast cancer. However, the underlying mechanism of its action has not been fully explored. In this study, we aimed to investigate the effect of sesamol on the transcriptome of MCF-7 breast cancer cells, in order to better understand its potential as an anti-cancer agent. The transcriptome profiles of MCF-7 breast cancer cells treated with sesamol were analyzed using Illumina deep-sequencing. The differentially expressed genes (DEGs) between the control and sesamol-treated groups were identified, and GO and KEGG pathway analyses of these DEGs were conducted using ClueGO. Protein-protein interaction (PPI) network of DEGs was mapped on STRING database and visualized by Cytoscape software. Hub genes in the network were screened by Cytohubba plugin of Cytoscape. Prognostic values of hub genes were analyses by the online Kaplan-Meier plotter and validated by qRT-PCR in MCF-7 cells. The results of the study showed that sesamol treatment had a significant effect on the transcriptome of MCF-7 cells, with a total of 351 DEGs identified. Functional enrichment analyses of DEGs revealed their involvement in extracellular matrix (ECM) remodeling, fatty acid metabolism and monocyte chemotaxis. The protein-protein interaction (PPI) network analysis of DEGs resulted in the identification of 10 hub genes, namely IGF2, MMP1, MSLN, CXCL10, WT1, ITGAL, PLD1, MME, TWIST1, and FOXA2. Survival analysis showed that MMP1 and ITGAL were significantly associated with overall survival (OS) and recovery-free survival (RFS) in breast cancer patients. Sesamol may play important roles in extracellular matrix (ECM) remodeling, fatty acid metabolism and cell cycle of MCF-7.

Abstract 532: Deficiency of CCN3 in Smooth Muscle Cells Perturbs Vascular Homeostasis and Exacerbates Thoracic Aortic Dissection

Introduction: Thoracic aortic dissection (TAD) is a life-threatening aortic disease with a high mortality rate in the event of rupture. Key mechanisms underlying the development of TAD include extracellular matrix (ECM) degradation and vascular smooth muscle cell (SMC) reprograming. Cellular communication network factor 3 (CCN3), a matricellular protein highly expressed in SMC, exerts a myriad of context-dependent biological functions. However, its role in SMC homeostasis and in the development of TAD remains largely undefined. Methods: A single-cell sequencing (Sc-seq) dataset (GSE213740) from human aortic dissection patients was analyzed to determine the association between CCN3 expression and TAD. In newly weaned male CCN3-floxed mice harboring Myh11-driven inducible Cre, 4 consecutive tamoxifen injections induced CCN3 deletion in SMCs. Mice without Cre expression were subjected to the same procedures and were used as controls. After 1 week of washout, 4-week-old mice were given 0.5% BAPN (β-aminopropionitrile monofumarate) in their drinking water to induce TAD. Histologic and protein measurements were performed on TAD tissues from control and CCN3-SMC-KO mice. Sc-seq analysis was performed to recapitulate cell heterogeneities and transcriptome profile changes. The regulatory role of SMC-derived CCN3 in TGFβ signaling was evaluated by western blot and colocalization analyses. Results: Analysis of the human Sc-seq dataset revealed downregulation of CCN3 in aortas from patients with TAD. CCN3-positive SMCs have elevated ECM and elastin gene expression in comparison to CCN3-null SMCs. In vivo, concordant with increased mortality in CCN3-SMC-KO animals, SMC-specific deletion of CCN3 promotes BAPN-induced elastin fibers fragmentation and degradation. In vitro, SMC deficiency of CCN3 diminished TGFβ-induced elastogenesis. CCN3 binds to the TGFβ receptor on SMCs and augments TGFβ receptor phosphorylation, thereby activating Smads, upregulating SMC elastogenesis, and as a consequence, alleviates elastin fiber fragmentation and TAD development. Conclusion: CCN3 is a key determinate of SMC reprogramming and sustains vascular homeostasis. Our findings suggest a novel target for the prevention and treatment of TAD.

Identifiation of novel effector regulatory T cells through simulaneous single cell epigenetic and transcriptomic profiling

Abstract Regulatory T cells (Tregs) harness a central role in peripheral immune tolerance. Since its discovery, there have been numerous efforts to dissect the role of Tregs in the context of disease condition. Recently, Treg fields are moving from the academia to industry since many evidences have been accumulated for the potential of treatment of inflammatory disease by administering genetically engineered Tregs. However, in the pre-clinical or clinical trials, this rare cell subset needs to be expanded in ex-vivo condition to achieve a sufficient number for the controlling of the disease. Our group previously established ex-vivo Treg expansion condition with 25 mer oligonucleotide to maintain the stability and function of Tregs. However, non-biased investigations on the perturbation of transcriptomic and epigenetic changes during the culture condition has not been explored. Since cutting edge multiomics tools which can simultaneously profile epigenetic and transcript signatures in a single cell resolution has been commercialized, we adopt 10x multiome technique to investigate our frozen ex-vivo expanded Tregs(eTreg) compared to freshly isolated frozen human Treg(fTreg) from the same donor. Compared to fTreg cells, we have identified a new subset that exists only in the eTreg cells and named it eeTreg. We found that the eeTreg resembles the phenotype of both effector Tregs and tumor-infiltrated Tregs. eeTreg is characterized by a significantly increased expression and chromatin accessibility score of FOXP3, STAT5B, ETS1 and RUNX1, which are known to be the essential transcription factors of regulatory T cell function. In summary, we suggest a novel human treg subset which might be manufactured by manipulating ex-vivo culture condition.

094 The impact of temporary or prolonged β-carotene deficiency during the transition period on the preovulatory granulosa cell transcriptomic profile at the time of breeding in dairy cows

094 The impact of temporary or prolonged β-carotene deficiency during the transition period on the preovulatory granulosa cell transcriptomic profile at the time of breeding in dairy cows

Abstract 632: Cell Populations In Pre-access Veins And Arteriovenous Fistulas: A Single Cell Perspective

Objective: A comprehensive picture of the cellular events dictating the transformation of the vein after arteriovenous fistula (AVF) creation has remained elusive so far. This study generated the first granular cellular atlas of pre-access veins and AVF from patients with end-stage kidney disease (ESKD). Methods: Veins (n=5) and AVF (n=7) were obtained from a total of 12 ESKD patients undergoing fistula creation in two stages. 41,000 enzymatically dissociated cells were single cell sequenced using the 10x Chromium system. Monocle3 inferred the trajectory leading to neointimal cells and signaling interactome in the AVF. Results: Cells populated 16 clusters containing canonical and non-canonical vascular markers after integrating the single cell transcriptomic profiles in Seurat v4. The majority of cells in both veins and AVF were smooth muscle cells (6.6 vs. 9.4%), fibroblasts (12.4 vs. 26.7%), neointimal-like cells (11.5% vs, 17.0, endothelial cells (10.5 vs. 10.5%), macrophages (26.1 vs. 19.5%), lymphocytes (16.4 vs.10.3), and neutrophils (0.7vs. 3.8 %). CENPF+ dividing cells were abundant in the AVF (0.2 to 1.2%). For the first time, we have detected a potential transcriptomic neointimal signature defined by the expression of BGN and PRELP genes that was increased in AVFs, but not absent in the pre-access veins, confirming previous histological findings. Neutrophilic infiltration (S100A8/9+ and CSF3R+) was increased in the AVF. Interestingly, lymphocytes and macrophages were decreased in the AVF with respect to the vein. These changes in cell populations in the AVF wall correlated with up-regulation of GO terms such as ECM deposition, neutrophil activation, and blood vessel remodeling in the AVF. Finally, we established a pseudotime cellular trajectory from contractile SMCs to neointimal-like cells, defined by COL8A1, PRELP, and BGN by Cohen’s I. Conclusions: We present a comprehensive single-cell atlas of the AVF transformation and assign identity to the cellular sources and cell trajectories in this remarkable vascular transformation.

Transcriptome and Proteome Profiling of Primary Human Gastric Interstitial Cells of Cajal Predicts Pacemaker Networks

Interstitial cells of Cajal (ICC) are specialized gastrointestinal (GI) pacemaker cells required for normal GI motility. Dysfunctions in ICC have been reported in patients with GI motility disorders, such as gastroparesis, who exhibit debilitating symptoms and greatly reduced quality of life. While the proteins, calcium-activated chloride channel anoctamin-1 (ANO1) and the receptor tyrosine kinase (KIT), are known to be expressed by human ICC, relatively little is known about the broad molecular circuitry underpinning human ICC functions. The present study therefore investigates the transcriptome and proteome of ANO1-expressing, KITlow/CD45-/CD11B- ICC obtained from primary human gastric tissue. Excess human gastric tissue resections were obtained from sleeve gastrectomy patients. ICC were purified using fluorescence-activated cell sorting (FACSorting). Then, ICC were characterized by using immunofluorescence, real-time polymerase chain reaction, RNA-sequencing and mass spectrometry. Compared to unsorted cells, real-time polymerase chain reaction showed the KITlow/CD45-/CD11B- ICC had: a 9-fold (P < 0.05) increase in ANO1 expression; unchanged KIT expression; and reduced expression for genes associated with hematopoietic cells (CD68, > 10-fold, P < 0.001) and smooth muscle cells (DES, > 4-fold, P < 0.05). RNA-sequencing and gene ontology analyses of the KITlow/CD45-/CD11B- cells revealed a transcriptional profile consistent with ICC function. Similarly, mass spectrometry analyses of the KITlow/CD45-/CD11B- cells presented a proteomic profile consistent with ICC activities. STRING-based protein interaction analyses using the RNA-sequencing and proteomic datasets predicted protein networks consistent with ICC-associated pacemaker activity and ion transport. These new and complementary datasets provide a valuable molecular framework for further understanding how ICC pacemaker activity regulates smooth muscle contraction in both normal GI tissue and GI motility disorders.

Non-contact immunological signaling for highly-efficient regulation of the transcriptional map of human monocytes.

The different immune system cells communicate and coordinate a response using a complex and evolved language of cytokines and chemokines. These cellular interactions carry out multiple functions in distinct cell types with numerous developmental outcomes. Despite the plethora of different cytokines and their cognate receptors, there is a restricted number of signal transducers and activators to control immune responses. Herein, we report on a new class of immunomodulatory signaling molecules based on volatile molecules (VMs, namely, volatile organic compounds [VOCs]), by which they can affect and/or control immune cell behavior and transcriptomic profile without any physical contact with other cells. The study demonstrates the role of VMs by analyzing non-contact cell communication between normal and cancerous lung cells and U937 monocytes, which are key players in the tumor microenvironment. Integrated transcriptome and proteome analyses showed the suggested regulatory role of VMs released from normal and cancer cells on neighboring monocytes in several molecular pathways, including PI3K/AKT, PPAR, and HIF-1. Presented data provide an initial platform for a new class of immunomodulatory molecules that can potentially mirror the genomic and proteomic profile of cells, thereby paving the way toward non-invasive immunomonitoring.

A Novel Gemcitabine-Resistant Gallbladder Cancer Model Provides Insights into Molecular Changes Occurring during Acquired Resistance.

Treatment options for advanced gallbladder cancer (GBC) are scarce and usually rely on cytotoxic chemotherapy, but the effectiveness of any regimen is limited and recurrence rates are high. Here, we investigated the molecular mechanisms of acquired resistance in GBC through the development and characterization of two gemcitabine-resistant GBC cell sublines (NOZ GemR and TGBC1 GemR). Morphological changes, cross-resistance, and migratory/invasive capabilities were evaluated. Then, microarray-based transcriptome profiling and quantitative SILAC-based phosphotyrosine proteomic analyses were performed to identify biological processes and signaling pathways dysregulated in gemcitabine-resistant GBC cells. The transcriptome profiling of parental and gemcitabine-resistant cells revealed the dysregulation of protein-coding genes that promote the enrichment of biological processes such as epithelial-to-mesenchymal transition and drug metabolism. On the other hand, the phosphoproteomics analysis of NOZ GemR identified aberrantly dysregulated signaling pathways in resistant cells as well as active kinases, such as ABL1, PDGFRA, and LYN, which could be novel therapeutic targets in GBC. Accordingly, NOZ GemR showed increased sensitivity toward the multikinase inhibitor dasatinib compared to parental cells. Our study describes transcriptome changes and altered signaling pathways occurring in gemcitabine-resistant GBC cells, which greatly expands our understanding of the underlying mechanisms of acquired drug resistance in GBC.

Transcriptome Analysis of Natural Killer Cells in Response to Newcastle Disease Virus Infected Hepatocellular Carcinoma Cells.

When tumor cells are infected by the Newcastle disease virus (NDV), the lysis of tumor cells by natural killer (NK) cells is enhanced, which may be related to the enhanced NK cell activation effect. To better understand the intracellular molecular mechanisms involved in NK cell activation, the transcriptome profiles of NK cells stimulated by NDV-infected hepatocellular carcinoma (HCC) cells (NDV group) and control (NC group, NK cells stimulated by HCC cells) were analyzed. In total, we identified 1568 differentially expressed genes (DEGs) in the NK cells of the NDV group compared to the control, including 1389 upregulated and 179 downregulated genes. Functional analysis showed that DEGs were enriched in the immune system, signal transmission, cell growth, cell death, and cancer pathways. Notably, 9 genes from the IFN family were specifically increased in NK cells upon NDV infection and identified as potential prognosis markers for patients with HCC. A qRT-PCR experiment was used to confirm the differential expression of IFNG and the other 8 important genes. The results of this study will improve our understanding of the molecular mechanisms of NK cell activation.

Abstract 2590: TP53 R249S mutation confers hepatic organoids with gain-of-function (GOF) tumorigenic features through transcriptional activation of ZMIZ2

Abstract Hepatocellular Carcinoma (HCC) represents the third leading cause of cancer-related mortality worldwide. TP53 mutations are pivotal genomic drivers for HCC development through frequent concurrent loss-of-function (LOF) aberrations and protein-altering missense mutations. Over 90% of the TP53 missense mutations are distributed within the core DNA binding domain (DBD), where R249S is the most common. Cumulative studies have demonstrated the cancer-promoting properties of TP53R249S mutation in HCC, but its biological impact on tumor initiation remains to be defined. To mimic the early stages of liver carcinogenesis, we generated normal hepatic organoids from human liver tissues of 3 individuals. CRISPR-Cas9 mediated knockout of TP53 in liver organoids resulted in pleomorphic malignant features, including dysplasia, hyperchromasia, atypical and frequent mitosis, loss of polarity, and increased nuclear to cytoplasm ratio. To recapitulate TP53R249S genotype in HCC, we ectopically expressed R249S mutant in liver organoids through lentiviral infection. TP53R249S liver organoids displayed tumorigenic properties as evidenced by increased lesion forming incidence (37.5%) in subcutaneous xenografts when compared with TP53KO (17%) and TP53 wild-type (WT) organoids (0%). Chromatin immunoprecipitation sequencing (ChIP-seq) analysis with HCC cell lines substantiated the specific gain-of-function (GOF) transcriptional activities of R249S mutant. A unique subset of transcription start site-proximal peaks was exclusively found in R249S mutant cells when compared with other TP53 missense mutants and WT. Integration of ChIP-seq and RNA-seq analysis identified transcription factor ZMIZ2 as a direct transcription target of R249S mutant. Our results showed that ZMIZ2 is preferentially overexpressed in HCC patients carrying TP53R249S mutation and exerts a vital role for proliferation of R249S mutant HCC cells. Knockdown of ZMIZ2 profoundly suppressed global H3K27 acetylation (H3K27ac) and H3K4 trimethylation (H3K4me3) in HCC cells. Transcriptome profiling of ZMIZ2 knockdown cells identified multiple downstream targets enriched in chromatin binding and interaction with histone deacetylase, further reinforcing its involvement in epigenetic regulation. In summary, our study revealed that TP53R249S mutation confers distinct advantages in increased tumorigenicity to human liver organoids through GOF transcriptional activities. ZMIZ2 serves as a direct downstream effector contributing to the oncogenic growth arising from R249S mutation plausibly through altering chromatin remodelling. Citation Format: Mingjing Xu, Yin Kau Lam, Jianqing Yu, Kelvin Kwok Chai Ng, Nathalie Wong. TP53 R249S mutation confers hepatic organoids with gain-of-function (GOF) tumorigenic features through transcriptional activation of ZMIZ2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2590.

Abstract 4195: Spatially resolved single cell transcriptomic profiling in formalin-fixed paraffin-embedded (FFPE) tissues

Abstract Formalin-fixed paraffin-embedded (FFPE) tissues are the most widely used clinical sample types in histology and molecular diagnosis, but these samples are often challenging for single-cell transcriptomic analysis due to RNA degradation and protein crosslinking. A spatial transcriptomics technique with high detection efficiency and single molecule resolution is required in order to accurately profile the gene expression in FFPE samples in situ. Vizgen’s MERSCOPE platform, built on multiplexed error robust in situ hybridization MERFISH technology, directly profiles intact tissue’s transcriptome with subcellular spatial resolution. Here, we demonstrate the FFPE MERSCOPE workflow in tissues from 10 mouse and human samples, including archival clinical samples. In each sample, hundreds of thousands of cells were captured with &amp;gt;100 million transcript counts, generating detailed spatial transcriptomic data for the profiled genes in each sample. A comparison of FFPE and matched fresh frozen samples indicated that the FFPE workflow performs similarly in detection efficiency as compared to the fresh frozen protocol. We further demonstrated the MERSCOPE FFPE workflow is compatible with protein imaging by performing simultaneous protein-based cell boundary staining with MERFISH to accurately profile gene expression and map cell types in archival clinical human samples. Finally, we constructed a spatially resolved single cell atlas across eight major tumor types, mapped and cataloged different cell types within the tumor microenvironment and systematically characterized the gene expression among cells. This study demonstrates the potential for spatially resolved transcriptomic profiling of FFPE samples at single cell level to contribute to a wide range of biomedical research areas, including many applications to study human diseases. Citation Format: Jiang He, Justin He, Timothy Wiggin, Rob Foreman, Renchao Chen, Nicolas Fernandez, George Emanuel. Spatially resolved single cell transcriptomic profiling in formalin-fixed paraffin-embedded (FFPE) tissues. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4195.

MRNA transcriptome profiling of human hepatocellular carcinoma cells HepG2 treated with Catharanthus roseus-silver nanoparticles.

The demand for the development of cancer nanomedicine has increased due to its great therapeutic value that can overcome the limitations of conventional cancer therapy. However, the presence of various bioactive compounds in crude plant extracts used for the synthesis of silver nanoparticles (AgNPs) makes its precise mechanisms of action unclear. To assessed the mRNA transcriptome profiling of human HepG2 cells exposed to Catharanthus roseus G. Don (C. roseus)-AgNPs. The proliferative activity of hepatocellular carcinoma (HepG2) and normal human liver (THLE3) cells treated with C. roseusAgNPs were measured using MTT assay. The RNA samples were extracted and sequenced using BGIseq500 platform. This is followed by data filtering, mapping, gene expression analysis, differentially expression genes analysis, Gene Ontology analysis, and pathway analysis. The mean IC50 values of C. roseusAgNPs on HepG2 was 4.38 ± 1.59 μg/mL while on THLE3 cells was 800 ± 1.55 μg/mL. Transcriptome profiling revealed an alteration of 296 genes. C. roseusAgNPs induced the expression of stress-associated genes such as MT, HSP and HMOX-1. Cellular signalling pathways were potentially activated through MAPK, TNF and TGF pathways that are responsible for apoptosis and cell cycle arrest. The alteration of ARF6, EHD2, FGFR3, RhoA, EEA1, VPS28, VPS25, and TSG101 indicated the uptake of C. roseus-AgNPs via both clathrin-dependent and clathrin-independent endocytosis. This study provides new insights into gene expression study of biosynthesised AgNPs on cancer cells. The cytotoxicity effect is mediated by the aberrant gene alteration, and more interestingly the unique selective antiproliferative properties indicate the C. roseusAgNPs as an ideal anticancer candidate.

Abstract OT2-19-01: Single cell characterization of longitudinal biopsies from breast cancer patients treated neoadjuvantly with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib in concert

Abstract Background The recent introduction of CDK4/6 inhibitors has been one of the most pivotal breakthroughs in breast cancer therapy during the last decades. A growing body of evidence proposes that CDK4/6 inhibitors influence the recruitment of immune cells in the tumor microenvironment with potential effects on the outcome. Study design The NeoLetRib-study is a multicenter, single-arm, open-label, neoadjuvant, phase II trial aiming at treating 100 locally advanced luminal-A and luminal-B breast cancer patients, defined as either large T2, or T3/T4, and/or N2-3. Patients receive neoadjuvant therapy for 6 months: ribociclib (600 mg daily, 21 days on/7 days off) and letrozole (2.5 mg daily). Peri- and premenopausal women also receive therapy with goserelin 3.6 mg s.c every 28 days. Methods Three sequencial tumor biopsies were collected: pre-treatment and on-treatment (cycle 1 - day 21 and cycle 6 - day 21 with ribociclib). These biopsies were subjected to single-cell transcriptome, T cell receptor and B cell receptor profiling using the Chromium Single-Cell v2 5′ Chemistry (10x Genomics). Libraries were paired-end sequenced on a NovaSeq6000. Single cell gene expression matrices were analyzed with the Seurat package (v4.0.2). After filtering out stressed/dying cells or cells with low quality sequencing, gene expression of the remaining good quality cells was normalized and scaled to construct principal components and further cluster cells. Results In this planned interim analysis, we clustered 242315 cells from longitudinal tumor biopsies from 18 patients at pre-treatment, 18 at cycle 1 - day 21 and 9 at cycle 6 - day 21, respectively. We identified 8 main cell types: T cells, B cells, epithelial cells, fibroblasts, endothelial cells, macrophages, mast cells and dendritic cells. To further identify specific and specialized cell subtypes, we clustered the cells belonging to the above-mentioned cell types and annotated the clusters obtained using validated marker genes. Statistical methods and algorithms were then used to characterize how the proportion of different cell types changes in tumors under treatment pressure. We identified significant changes in immune cell proportions, including regulatory T cells, CD14 monocytes, SLC2A1-Macrophages among others. Conclusions In this unique patient cohort, we used single cell transcriptome profiling to obtain a high-resolution map of cell types found in tumor biopsies from the NeoLetRib trial. We characterized the effects of the combination of ribociclib and letrozole on the tumor microenvironment and identified cells sensitive and resistant to treatment. In this interim analysis, the observed longitudinal changes of immune cell types proportions in the tumor microenvironment might suggest immune related effects of the treatment combination. Citation Format: Marie Fongård, Chloé Steen, Salim Ghannoum, Marius Bjørnstad, Barbro Holm, Tatjana Bosnjak, Laurens Reitsma, Stephanie Geisler, Kamilla Fjermeros, Johannes Bruteig, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Marie Loeng, Aino Vuoriluoto, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli. Single cell characterization of longitudinal biopsies from breast cancer patients treated neoadjuvantly with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib in concert [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-19-01.

Thyroid cells from normal and autoimmune thyroid glands suppress T lymphocytes proliferation upon contact revealing a new regulatory inhibitory type of interaction independent of PD1/PDL1

Immune Checkpoint Receptors include a number of inhibitory receptors that limit tissue damage during immune responses; blocking PD-1/PD-L1 checkpoint receptor axis led to a paradigm shift in cancer immunotherapy but also to autoimmune adverse effects, prominently thyroid autoimmunity. Although PD-L1 is known to be expressed on thyroid follicular cells (TFCs) of autoimmune glands the role on PD-1/PD-L1 in the interaction between T cells and thyroid cells in the tissue has not been investigated. Here we report that autologous primary TFCs, but not transformed TFCs, inhibit CD4 and CD8 T cell proliferation but no cytokine production. This effect is not, however, mediated by PD-1/PD-L1 nor locally produced cytokines. Beta galactosidase analysis excluded culture-induced senescence as an explanation. High resolution flow cytometry demonstrated that autologous TFC/T cells co-culture induced the expansion of several clusters of double negative (DN) T cells characterized by high expression of activation markers and negative immune checkpoints. Single cell transcriptomic profiling demonstrated that dissociated TFC express numerous candidate molecules for mediating this suppressive activity, including CD40, E-Cadherin and TIGIT ligands. These ligands directly or through the generation of a suppressor population of DN T cells, and not the PD-1/PD-L1 axis, are most likely the responsible of TFC immunosuppressive activity. These results contribute to reveal the complex network of inhibitory mechanism that operate at the tissue level to restrain autoimmunity but also point to pathways, other that PD-1/PD-L1, that can contribute to tumor evasion.

Single-Cell RNA Transcriptome Profiling of Liver Cells of Short-Term Alcoholic Liver Injury in Mice

Alcoholic liver disease (ALD) is currently considered a global healthcare problem with limited pharmacological treatment options. There are abundant cell types in the liver, such as hepatocytes, endothelial cells, Kupffer cells and so on, but little is known about which kind of liver cells play the most important role in the process of ALD. To obtain a cellular resolution of alcoholic liver injury pathogenesis, 51,619 liver single-cell transcriptomes (scRNA-seq) with different alcohol consumption durations were investigated, 12 liver cell types were identified, and the cellular and molecular mechanisms of the alcoholic liver injury were revealed. We found that more aberrantly differential expressed genes (DEGs) were present in hepatocytes, endothelial cells, and Kupffer cells than in other cell types in alcoholic treatment mice. Alcohol promoted the pathological processes of liver injury; the specific mechanisms involved: lipid metabolism, oxidative stress, hypoxia, complementation and anticoagulation, and hepatocyte energy metabolism on hepatocytes; NO production, immune regulation, epithelial and cell migration on endothelial cells; antigen presentation and energy metabolism on Kupffer cells, based on the GO analysis. In addition, our results showed that some transcription factors (TFs) are activated in alcohol-treated mice. In conclusion, our study improves the understanding of liver cell heterogeneity in alcohol-fed mice at the single-cell level. It has potential value for understanding key molecular mechanisms and improving current prevention and treatment strategies for short-term alcoholic liver injury.

Bioinformatics analysis of the common targets of miR-223-3p, miR-122-5p, and miR-93-5p in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is one of the most common gynecological endocrine disorders. MicroRNAs (miRNAs) play extensive roles in the pathogenesis of PCOS and can serve as potential diagnostic markers. However, most studies focused on the regulatory mechanisms of individual miRNAs, and the combined regulatory effects of multiple miRNAs remain unclear. The aim of this study was to identify the common targets of miR-223-3p, miR-122-5p, and miR-93-5p; and assess the transcript levels of some of these targets in PCOS rat ovaries. Transcriptome profiles of granulosa cells from PCOS patients were obtained from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs). A total of 1,144 DEGs were screened, 204 of which were upregulated and 940 were downregulated. According to the miRWalk algorithm, 4,284 genes were targeted by all three miRNAs at the same time, and intersection with DEGs was used to obtain candidate target genes. A total of 265 candidate target genes were screened, and the detected target genes were subjected to Gene ontology (GO) and KEGG pathway enrichment, followed by PPI network analysis. Then, qRT-PCR was used to determine the levels of 12 genes in PCOS rat ovaries. The expressions of 10 of these genes were found to be consistent with our bioinformatics results. In conclusion, JMJD1C, PLCG2, SMAD3, FOSL2, TGFB1, TRIB1, GAS7, TRIM25, NFYA, and CALCRL may participate in the development of PCOS. Our findings contribute to the identification of biomarkers that may promote the effective prevention and treatment of PCOS in the future.

Long-read sequencing reveals the effect of follicle-stimulating hormone on the mRNA profile of chicken granulosa cells from prehierarchical follicles

Follicle selection is an important step in the laying process of chicken, which is closely related to the laying performance and fecundity of hens. Follicle selection mainly depends on the regulation of follicle-stimulating hormone (FSH) secreted by pituitary gland and the expression of follicle stimulation hormone receptor. To uncover the role of FSH in chicken follicle selection, in this study, we analyzed the changes in the mRNA transcriptome profiles of FSH-treated chicken granulosa cells from prehierarchical follicles by long-read sequencing Oxford Nanopore Technologies (ONT) approach. Among the 10,764 genes detected, 31 differentially expressed (DE) transcripts of 28 DE genes were significantly upregulated by FSH treatment. These DE transcripts (DETs) were mainly related to the steroid biosynthetic process by GO analysis and enriched in pathways of ovarian steroidogenesis and aldosterone synthesis and secretion by KEGG analysis. Among these genes, the mRNA and protein expression of TNF receptor associated factor 7 (TRAF7) was upregulated after FSH treatment. Further study revealed that TRAF7 stimulated the mRNA expression of steroidogenic enzymes steroidogenic acute regulatory protein (StAR) and cytochrome P450 family 11 subfamily A member 1 (CYP11A1) genes and the proliferation of granulosa cells. This is the first study to investigate differences in chicken prehierarchical follicular granulosa cells before and after FSH treatment by using ONT transcriptome sequencing, which provides a reference for a more comprehensive understanding of the molecular mechanism of follicle selection in chicken.

Transcriptome of GH-producing pituitary neuroendocrine tumours and models are significantly affected by somatostatin analogues

Pituitary neuroendocrine tumours (PitNETs) are neoplasms of the pituitary that overproduce hormones or cause unspecific symptoms due to mass effect. Growth hormone overproducing GH-producing PitNETs cause acromegaly leading to connective tissue, metabolic or oncologic disorders. The medical treatment of acromegaly is somatostatin analogues (SSA) in specific cases combined with dopamine agonists (DA), but almost half of patients display partial or full SSA resistance and potential causes of this are unknown. In this study we investigated transcriptomic landscape of GH-producing PitNETs on several levels and functional models—tumour tissue of patients with and without SSA preoperative treatment, tumour derived pituispheres and GH3 cell line incubated with SSA to study effect of medication on gene expression. MGI sequencing platform was used to sequence total RNA from PitNET tissue, pituispheres, mesenchymal stromal stem-like cells (MSC), and GH3 cell cultures, and data were analysed with Salmon—DeSeq2 pipeline. We observed that the GH-producing PitNETs have distinct changes in growth hormone related pathways related to its functional status alongside inner cell signalling, ion transport, cell adhesion and extracellular matrix characteristic patterns. In pituispheres model, treatment regimens (octreotide and cabergoline) affect specific cell proliferation (MKI67) and core functionality pathways (RYR2, COL8A2, HLA-G, ARFGAP1, TGFBR2). In GH3 cells we observed that medication did not have transcriptomic effects similar to preoperative treatment in PitNET tissue or pituisphere model. This study highlights the importance of correct model system selection for cell transcriptomic profiling and data interpretation that could be achieved in future by incorporating NGS methods and detailed cell omics profiling in PitNET model research.

Transcriptome profiling of bovine endometrial epithelial cells induced by lipopolysaccharides in vitro

Endometritis is an inflammation of the surface of the endometrium that does not penetrate the submucosa and can cause infertility and increase the elimination rate in cows. Endometrial epithelial cells are the first barrier of the endometrium against foreign stimuli and bacterial infection. Understanding the genetic changes in stimulated endometrial epithelial cells will help in the efforts to prevent and treat endometritis. This study investigated changes in bovine endometrial epithelial (BEEC) gene expression induced by lipopolysaccharide (LPS)-induced inflammation and compared transcriptome-wide gene changes between LPS- and phosphate-buffered saline (PBS)- treated BEECs by RNA sequencing. Compared with the PBS group, the LPS group showed 60 differentially expressed genes (DEGs) (36 upregulated, 24 downregulated). Gene Ontology enrichment analysis revealed that most enrichment occurred during CXCR chemokine receptor binding, inflammatory response, and neutrophil migration. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed DEGs mainly concentrated in cytokine–cytokine receptor interactions; IL-17, tumor necrosis factor, NOD-like receptor, chemokine, Toll-like receptor, and nuclear factor-κB signaling pathways; and the cytoplasmic DNA sensing pathway. Moreover, results revealed that cytokines SAA3 and HP increased significantly after LPS treatment. These effects of LPS on BEECs transcriptome and the molecular mechanism of endometritis provide a basis for improved clinical treatment and novel drug development.

Comparative Transcriptome Profiles of Human HaCaT Cells in Response to Gynostemma pentaphyllum Extracts Obtained Using Three Independent Methods by RNA Sequencing.

Gynostemma pentaphyllum (GP) is widely used in herbal medicine. In this study, we developed a method for the large-scale production of GP cells using plant tissue culture techniques combined with bioreactors. Six metabolites (uridine, adenosine, guanosine, tyrosine, phenylalanine, and tryptophan) were identified in GP extracts. Transcriptome analyses of HaCaT cells treated with GP extracts using three independent methods were conducted. Most differentially expressed genes (DEGs) from the GP-all condition (combination of three GP extracts) showed similar gene expression on treatment with the three individual GP extracts. The most significantly upregulated gene was LTBP1. Additionally, 125 and 51 genes were upregulated and downregulated, respectively, in response to the GP extracts. The upregulated genes were associated with the response to growth factors and heart development. Some of these genes encode components of elastic fibers and the extracellular matrix and are associated with many cancers. Genes related to folate biosynthesis and vitamin D metabolism were also upregulated. In contrast, many downregulated genes were associated with cell adhesion. Moreover, many DEGs were targeted to the synaptic and neuronal projections. Our study has revealed the functional mechanisms of GP extracts' anti-aging and photoprotective effects on the skin using RNA sequencing.