Gene expression profiling of the bone trabecula in patients with osteonecrosis of the femoral head by RNA sequencing

2019 ◽  
Vol 166 (6) ◽  
pp. 475-484
Author(s):  
Haobo Bai ◽  
Tingmei Chen ◽  
Qian Lu ◽  
Weiwen Zhu ◽  
Jian Zhang
Keyword(s):  
Gene Expression ◽  
Femoral Head ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Enrichment Analysis ◽  
Control Group ◽  
Pathway Enrichment Analysis ◽  
Bone Trabecula ◽  
Mrna Profiling ◽  
Normal Bone

Abstract Early diagnosis and treatment of osteonecrosis of the femoral head (ONFH) is challenging. Bone trabecula play a vital role in the severity and progression of ONFH. In the present study, the investigators used gene expression profiling of bone trabecula to investigate gene alterations in ONFH patients. Osteonecrotic bone trabecula (ONBT) such as necrosis, fibrosis, and lacuna were confirmed by histological examination in the patients. The adjacent ‘normal’ bone trabecula (ANBT) did not show any pathological changes. Gene sequencing data revealed that although ANBT showed no significant histological changes, alteration of mRNA profiling in ANBT was observed, similar to that in ONBT. Our results indicated that the alteration of mRNA profiling in ANBT may cause normal bone tissue to develop into necrotic bone. RNA-seq data indicated that 2,297 differentially abundant mRNAs were found in the ONBT group (1,032 upregulated and 1,265 downregulated) and 1,523 differentially abundant mRNAs in the ANBT group (744 upregulated and 799 downregulated) compared with the healthy control group. Gene ontology (GO) enrichment analysis suggested that fatty acid metabolism and degradation were the main zones enriched with differentially expressed genes (DEG). Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis indicated that peroxisome proliferator-activated receptor γ (PPAR-γ) pathway was the most significantly regulated pathway. Lipocalin-2 (LCN2), an osteoblast-enriched secreted protein, was significantly decreased in ONBT suggesting that downregulation of LCN2 might affect lipid metabolism and lead to hyperlipidemia, and thus promote pathogenesis of ONFH.

scholarly journals Identification and Validation of Hub Genes Related to Meniscus Senescence Based on Gene Expression Profiling Analysis

2021 ◽  
Author(s):  
ming chen ◽  
Siqi Zhou ◽  
Huasong Shi ◽  
Hanwen Gu ◽  
Yinxian Wen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Enrichment Analysis ◽  
Functional Enrichment ◽  
Pathway Enrichment Analysis ◽  
Hub Genes ◽  
Meniscus Cells ◽  
Toxic Drugs ◽  
Profiling Analysis

Abstract Background: The incidence of meniscal injury is on the rise, partly due to the general aging of the population. The compositional change in the meniscus with aging would increase the tissue vulnerability of the meniscus, which would induce meniscus tearing. Here, we investigated the molecular mechanism of age-related meniscus degeneration with gene expression profiling analysis.Methods: The GSE45233 dataset, including 6 elderly meniscus samples and 6 younger meniscus samples, which were obtained from patients undergoing arthroscopic partial meniscectomy, was downloaded from the Gene Expression Omnibus (GEO) database for subsequent bioinformatics analysis. To screen the differential expression of mRNAs, identify the miRNAs targeting hub genes, and forecast the potentially toxic drugs, we completed a series of bioinformatics analyses, including functional and pathway enrichment analysis, protein-protein interaction network, hub genes screening, construction of a lncRNA–miRNA–mRNA network, and molecular docking of potential drugs. Furthermore, hub genes were examined in human senescent menisci, mouse senescent meniscus tissues and mouse meniscus cells stimulated by IL-1β.Results: In total, the most significant 4 hub genes (RRM2, AURKB, CDK1, and TIMP1), 5 miRNAs (hsa-miR-6810-5p, hsa-miR-4676-5p, hsa-miR-6877-5p, hsa-miR-8085, and hsa-miR-6133) that could regulate such 4 hub genes and potential toxic drugs (Cladribine, Danusertib, Barasertib, Riviciclib, and Dinaciclib) that may have a targeting effect on these genes, were finally identified. The functional enrichment results showed that hub genes were mainly concentrated in aging and regulation of the cell cycle process. Further pathways enrichment analysis of these miRNA revealed that these miRNAs were involved in the synthesis of glycosaminoglycans. The hub genes were decreased in meniscus cells in vitro and meniscus tissues in vivo, which indicated that hub genes were related to meniscus senescence.Conclusions: In a word, our current study would provide a basis for finding markers of the aging meniscus to a certain extent.

scholarly journals The Carcinogenome Project: In-vitro Gene Expression Profiling of Chemical Perturbations to Predict Long-Term Carcinogenicity

2018 ◽  
Author(s):  
Amy Li ◽  
Xiaodong Lu ◽  
Ted Natoli ◽  
Joshua Bittker ◽  
Nisha Sipes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Enrichment Analysis ◽  
Pathway Enrichment Analysis ◽  
Pathway Enrichment ◽  
Link Type ◽  
Chemical Perturbations

AbstractBackground: Most chemicals in commerce have not been evaluated for their carcinogenic potential. The current de-facto gold-standard approach to carcinogen testing adopts the two-year rodent bioassay, a time consuming and costly procedure. Alternative approaches, such as high-throughput in-vitro assays, show promise in addressing the limitations in carcinogen screening.Objectives: We developed a screening process for predicting chemical carcinogenicity and genotoxicity and characterizing modes of actions (MoAs) using in-vitro gene expression assays.Methods: We generated a large toxicogenomics resource comprising ~6,000 expression profiles corresponding to 330 chemicals profiled in HepG2 cells at multiple doses and in replicates. Predictive models of carcinogenicity were built using a Random Forest classifier. Differential pathway enrichment analysis was performed to identify pathways associated with carcinogen exposure. Signatures of carcinogenicity and genotoxicity were compared with external data sources including Drugmatrix and the Connectivity Map.Results: Among profiles with sufficient bioactivity, our classifiers achieved 72.2% AUC for predicting carcinogenicity and 82.3% AUC for predicting genotoxicity. Our analysis showed that chemical bioactivity, as measured by the strength and reproducibility of the transcriptional response, is not significantly associated with long-term carcinogenicity, as evidenced by the many carcinogenic chemicals that did not elicit substantial changes in gene expression at doses up to 40 μM. However, sufficiently high transcriptional bioactivity is necessary for a chemical to be used for prediction of carcinogenicity. Pathway enrichment analysis revealed several pathways consistent with literature review of pathways that drive cancer, including DNA damage and DNA repair. These data are available for download via https://clue.io/CRCGN_ABC, and a web portal for interactive query and visualization of the data and results is accessible at https://carcinogenome.org.Conclusions: We demonstrated a short-term in-vitro screening approach using gene expression profiling to predict long-term carcinogenicity and infer MoAs of chemical perturbations.

scholarly journals Innate inflammatory gene expression profiling in potential brain-dead donors: detailed investigation of the effect of common corticosteroid therapy

2017 ◽  
Vol 23 (5) ◽  
pp. 440-448 ◽  
Author(s):  
Reza Gholamnezhadjafari ◽  
Nader Tajik ◽  
Reza Falak ◽  
Reza Aflatoonian ◽  
Sanaz Dehghan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Inflammatory Factors ◽  
Control Group ◽  
Organ Donors ◽  
Complement Components ◽  
Tnf Α ◽  
Brain Dead

Our study aimed to assess the influence of common methylprednisolone therapy on innate inflammatory factors in potential brain-dead organ donors (BDDs). The study groups consisted of 50 potential BDDs who received 15 mg/kg/d methylprednisolone and 25 live organ donors (LDs) as control group. Innate immunity gene expression profiling was performed by RT-PCR array. Soluble serum cytokines and chemokines, complement components, heat shock protein 70 (HSP70) and high mobility group box-1 (HMGB1) were measured by ELISA. Surface expression of TLR2 and TLR4 were determined using flow cytometry. Gene expression profiling revealed up-regulation of TLRs 1, 2, 4, 5, 6, 7 and 8, MYD88, NF-κB, NF-κB1A, IRAK1, STAT3, JAK2, TNF-α, IL-1β, CD86 and CD14 in the BDD group. Remarkably, the serum levels of C-reactive protein and HSP70 were considerably higher in the BDD group. In addition, serum amounts of IL-1β, IL-6, TNF-α, HMGB1, HSP70, C3a and C5a, but not IL-8, sCD86 or monocyte chemoattractant protein-1, were significantly increased in the BDD group. Significant differences were observed in flow cytometry analysis of TLR2 and TLR4 between the two groups. In summary, common methylprednisolone therapy in BDDs did not adequately reduce systemic inflammation, which could be due to inadequate doses or inefficient impact on other inflammatory-inducing pathways, for example oxidative stress or production of damage-associated molecules.

Intraplatform Reproducibility and Technical Precision of Gene Expression Profiling in Four Laboratories Investigating 112 Leukemia Samples: The DACH Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2383-2383
Author(s):  
Alexander Kohlmann ◽  
Elisabeth Haschke-Becher ◽  
Barbara Wimmer ◽  
Ariana Huber-Wechselberger ◽  
Sandrine Meyer-Monard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Control Group ◽  
Unsupervised Hierarchical Cluster ◽  
Daily Routine ◽  
Total Rna

Abstract Gene expression profiling has the potential to offer consistent objective diagnostic test results once a standardized protocol is established. We investigated the robustness, precision, and reproducibility of this technology and present data that complements the Microarray Innovations in LEukemia study (MILE study). In four laboratories, located in Germany (D), Austria (A), and Switzerland (CH) (DACH study), replicates of 112 patient samples were analyzed using the AmpliChip Leukemia research test. Patient samples were centrally collected and diagnosed in daily routine at the Munich Leukemia Laboratory and represented 8 distinct classes of acute and chronic leukemias, with non-leukemia as control group. After purification of the mononuclear cells by Ficoll density centrifugation, 4 × 5 million cells were frozen in lysis buffer and stored at −80°C. Equipped with identical instruments, software, and reagents, study operators were trained on the microarray sample preparation protocol using total RNA from commercially available cell lines. Upon receipt of the frozen lysates each of the four laboratories purified the total RNA from the 112 technical quadruplicates. 99.3% (445/448) of the sample preparations were successfully performed. On average, 8.4 μg, 7.2 μg, 7.4 μg, or 7.5 μg of total RNA, respectively, were isolated from the mononuclear cells from the four laboratories. In three samples less than 1.0 μg of total RNA was obtained and thus the preparation failed. Bland-Altman plots of agreement showed that any two centers were unlikely to have more than an 8.3 μg difference in yield of total RNA from the same sample. On average there was between 0.1 μg to 1.2 μg difference in total RNA yield from the same sample. Further processing of the 445 samples resulted in 437 (98.2%) successfully performed in vitro transcription reactions, i.e. obtained cRNA yield of >8.0 μg. On average there was between 0.4 μg to 7.4 μg difference in cRNA yield from the same sample. After hybridization to microarrays on average, 46.1%, 48.6%, 46.5%, and 47.3% of probe sets were detected as present with mean scaling factors of 4.3, 2.9, 3.9, and 3.7, respectively. The mean values and standard deviations of distributions of the coefficient of variation (CV) within each site over all the probe sets of the quantile normalized signals on the chip were 27.2% (StdDev: 12.3%), 27.0% (StdDev: 12.3%), 27.3% (StdDev: 12.3%), 26.9% (StdDev: 12.4%), respectively. Furthermore, in unsupervised hierarchical cluster and principal component analyses replicates from the same patient always clustered closely together, with no indications of association between gene expression profiles due to different operators or laboratories. In conclusion, we demonstrated that microarray analysis can be performed with remarkably high inter-laboratory reproducibility and with comparable quality and high technical precision across laboratories.

Characterization of Mesenchymal Stromal Cells (MSCs) Derived From Acute Myeloid Leukemia (AML) Bone Marrow

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2558-2558
Author(s):  
Soumit K. Basu ◽  
Xin Zhao ◽  
Sylvia Chien ◽  
Min Fang ◽  
Vivian Oehler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Stromal Cell ◽  
Collagen Type ◽  
Β1 Integrin ◽  
Normal Bone Marrow ◽  
Bone Marrow Mscs ◽  
Normal Bone

Abstract Abstract 2558 INTRODUCTION Recent evidence has implicated the bone marrow microenvironment directly in the pathogenesis of preleukemic bone marrow disorders (Raaijmakers MHGP et al, Nature 2010) with potential to transform to AML. Moreover, the bone marrow microenvironment is critical to AML survival (Garrido et al 2001, Meads et al 2008). We sought to investigate aspects of the bone marrow microenvironment which may contribute to the pathogenesis and persistence of AML by direct analysis of primary bone marrow MSCs isolated from AML patients in comparison with primary bone marrow MSCs from normal subjects. Our analyses included (1) a comparison of cytokine elaboration between normal and AML bone marrow MSCs (2) immunophenotyping of normal and AML bone marrow MSCs (3) characterisation of binding by AML cells to their autologous stroma and (4) gene expression profiling of normal and AML bone marrow MSCs and (5) cytogenetic analysis AML bone marrow MSCs. METHODS We have been able to derive confluent cultures of mesenchymal stromal cells from 80% of AML patient marrow samples. Fresh or cryopreserved bone marrow samples were plated in non-hematopoietic expansion media (Miltenyi) under reduced oxygen conditions. After 48 hours of culture, nonadherent cells are removed, and over a period of 1–2 weeks, cultures of spindle shaped cells are derived, that can be sustained in culture for up to 5 passages. Similar cultures were derived from normal bone marrow. Gene expression profiling of bone marrow MSCs was performed by whole genome analysis using Illumina's BeadChip microarray platform. Samples included mRNAs isolated from confluent cultures of AML bone marrow MSCs, normal bone marrow MSCs, and the normal bone marrow stromal cell lines HS27a and HS5. RESULTS Comparison of cytokine elaboration showed a statistically significant (p = 0.037) 5-fold decrease in stromal MCP-1 production by AML bone marrow MSCs compared with normal bone marrow MSCs (327 ± 169 vs. 1669 ± 570 pg/mL, mean ± SE). Normal and AML MSCs showed no statistically significant differences in the production of G-CSF, GM-CSF, M-CSF, IL6, IL12, SCF, TNFα, MCP1 and SDF1β. Like their normal counterparts, AML bone marrow MSCs strongly express CD90, CD29 (β1 integrin), CD73, CD105, CD146, and CD44. The normal bone marrow derived stromal cell lines HS27a and HS5 demonstrated moderate expression of CD324/E-cadherin (28.4% and 37.9% respectively). E-cadherin expression proved highly variable among normal bone marrow MSCs (1.9%-54.9%) and similarly variable in AML bone marrow MSCs (7.8%–56.5%). AML binding to autologous MSCs primarily dependent on β1 integrin, L-selectin and VCAM-1. In contrast, prior data (Basu et al., ASH 2010 Abstract 2756) demonstrated AML binding to the normal bone marrow stromal cell line HS27a as primarily dependent on β1 integrin, CXCR4, and E-cadherin. Gene expression profiling demonstrated no significant differences between 6 AML and 5 normal bone marrow MSCs. AML bone marrow MSCs, as expected, demonstrated marked differences from AML bone marrow mononuclear cells, expressing higher levels of connective tissue growth factor (128 fold), tropomyosin 1 (84.4 fold), collagen type 1 α1 (194 fold), collagen type 1 α2 (137 fold), collagen type 4 α1 (128 fold), collagen type 5 α1 (119 fold), transgelin (111 fold), cadherin 11 (21 fold), biglycan (137 fold), IGF binding protein 6 (18 fold), and procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (74 fold). In our cytogenetic analyses of MSCs to date, bone marrow MSCs derived from one of the complex karyotype AML patients demonstrated normal cytogenetics. In contrast, bone marrow MSCs derived from a second AML patient shared a common t(2;11) translocation present in the AML cells but demonstrated an abnormal clone with del(4q) which lacked the t(6;9) also present in the AML cells [i.e.-MSC karyotype: t(2;11), del(4q); AML karyotype: t(2;11), t(6;9)]. These results suggest that in some patients AML cells and their autologous MSCs may share the same clonal origin, while in other cases, the MSCs may have a distinct origin. CONCLUSION AML and normal bone marrow MSCs demonstrate only subtle differences, providing an explanation of the ability of AML bone marrow MSCs to support normal hematopoiesis after leukemic debulking (e.g. via induction chemotherapy or allogeneic stem cell transplant). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Integrative analysis of transcriptome-wide association study and gene expression profiling identifies candidate genes associated with stroke

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7435
Author(s):  
Jian Yang ◽  
Bin Yan ◽  
Yajuan Fan ◽  
Lihong Yang ◽  
Binbin Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Candidate Genes ◽  
Expression Profiling ◽  
Enrichment Analysis ◽  
Integrative Analysis ◽  
Functional Gene ◽  
Gene Sets ◽  
Go Enrichment ◽  
Go Enrichment Analysis

Background Stroke is a major public health burden worldwide. Although genetic variation is known to play a role in the pathogenesis of stroke, the specific pathogenic mechanisms are still unclear. Transcriptome-wide association studies (TWAS) is a powerful approach to prioritize candidate risk genes underlying complex traits. However, this approach has not been applied in stroke. Methods We conducted an integrative analysis of TWAS using data from the MEGASTROKE Consortium and gene expression profiling to identify candidate genes for the pathogenesis of stroke. Gene ontology (GO) enrichment analysis was also conducted to detect functional gene sets. Results The TWAS identified 515 transcriptome-wide significant tissue-specific genes, among which SLC25A44 (P = 5.46E−10) and LRCH1 (P = 1.54E−6) were significant by Bonferroni test for stroke. After validation with gene expression profiling, 19 unique genes were recognized. GO enrichment analysis identified eight significant GO functional gene sets, including regulation of cell shape (P = 0.0059), face morphogenesis (P = 0.0247), and positive regulation of ATPase activity (P = 0.0256). Conclusions Our study identified multiple stroke-associated genes and gene sets, and this analysis provided novel insights into the genetic mechanisms underlying stroke.

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