Genome-Wide Transcriptome Profiling of the Neoplastic Giant Cell Tumor of Bone Stromal Cells by RNA Sequencing.

Giant cell tumor of bone (GCTB) is the most common non-malignant primary bone tumor reported in Hong Kong. Failure of treatment in advanced GCTB with aggressive local recurrence remains a clinical challenge. In order to reveal the molecular mechanism underlying the pathogenesis of this tumor, we aimed to examine the transcriptome profiling of the neoplastic stromal cells of GCTB in this study. RNA-sequencing was performed on three GCTB stromal cell samples and one bone marrow-derived MSC sample and 174 differentially expressed genes (DEGs) were identified between these two cell types. The top five up-regulated genes are SPP1, F3, TSPAN12, MMP13, and LGALS3BP and further validated by qPCR and Western Blotting. Knockdown of SPP1 was found to induce RUNX2 and OPG expression in GCTB stromal cells but not the MSCs. Ingenuity pathway analysis (IPA) of the 174 DEGs revealed significant alternations in 23 pathways; variant calling analysis revealed 1915 somatic variants of 384 genes with high or moderate impacts. Interestingly, four canonical pathways were found overlapping in both analyses; from which VEGFA, CSF1, PLAUR, and F3 genes with somatic mutation were found up-regulated in GCTB stromal cells. The STRING diagram showed two main clusters of the DEGs; one cluster of histone genes that are down-regulated in GCTB samples and another related to osteoblast differentiation, angiogenesis, cell cycle progression, and tumor growth. The DEGs and somatic mutations found in our study warrant further investigation and validation, nevertheless, our study add new insights in the search for new therapeutic targets in treating GCTB. J. Cell. Biochem. 118: 1349-1360, 2017. © 2016 Wiley Periodicals, Inc.

Profiling of the silica-induced molecular events in lung epithelial cells using the RNA-Seq approach.

Silicosis is a prolonged, irreversible and incurable occupational disease, and there is a significant number of newly diagnosed cases every year in Hong Kong. Due to the long latency of the disease, the diagnosis can be missed until detailed clinical examination at a later stage. For a better control of this deadly disease, detailing the pro-inflammatory and fibrotic events in the macrophage would be instrumental in understanding the pathogenesis of the disease and essential for the significant biomarkers discovery. In this in vitro study, human cell line model A549 lung epithelial cells were used. The immediate molecular events underneath the activation of quartz silica polymorphs were followed in a time course of 0, 0.5, 2, 8, 16 and 24 h. The transcriptome library was prepared and subjected to RNA-Seq analysis. Data analysis was performed by pathway analysis tools and verified by real-time PCR. The results showed that triggered genes were mainly found in the immune response and inflammatory pathways. An interesting finding was the association of the DNA-binding protein inhibitor (ID) family in the silica exposure to lung cells. The linkage of ID1, ID2 and ID3 to cancer may rationalize themselves to be the markers indicating an early response of silicosis. However, further studies are required to consolidate the roles of these genes in silicosis. Copyright © 2017 John Wiley & Sons, Ltd.

RNA-Seq revealed ATF3-regulated inflammation induced by silica.

BACKGROUND: Millions of workers are exposed to dust containing silica. Chronic and over-exposure to silica will lead to silicosis, which is an irreversible and sometimes fatal lung disease. The disordered physiological processes of silicosis consist of accumulation of silica particles in the alveoli of the lung. Then, the ingestion of the silica particles by macrophages was followed by an inflammatory response. Up till now, the chest radiographs remain the key tool in diagnosing and assessing the extent of silicosis. However, concerns exist regarding the sensitivity and specificity of the technique. Therefore, there is still a need to develop a biomarker for silicosis for early detection of silicosis. METHOD: In this study, RNA-Seq was applied to detect the gene expression changes when silica was exposed to macrophages at different time intervals. RNA-Seq provides a broader dynamic range, increased specificity and sensitivity, and easier detection of rare and low-abundance transcripts. Bioinformatics tools such as the Database for Annotation, Visualization and Integrated Discovery (DAVID) and Gene Functional Classification Tool and Search Tool for the Retrieval of Interacting Genes (STRING) were applied for data analysis. Quantitative PCR was used to validate the results. RESULTS: Our results showed that regulation of transcription factors was the dominant activated pathway in early exposure of silica to macrophages, followed by inflammatory responses which were the main mechanisms in silicosis. One of the findings was the upregulation of activating transcription factor 3 (ATF3) during silica exposure. When ATF3 expression was inhibited by siRNA, the production of cytokines IL-1ß, IL-6 and TNF was further increased. CONCLUSION: This indicated that ATF3 may be a potential early diagnostic biomarker for silicosis and ATF3 acts as a repressor in inflammatory responses induced by silica.