Screening and bioinformatic analysis of differentially expressed miRNAs in serum of patients with pneumoconiosis / 中国职业医学

OBJECTIVE: To screen the differentially expressed microRNA(miRNA) in the serum of patients with occupational pneumoconiosis(hereinafter referred to as pneumoconiosis), and explore their potential target genes and related transcription factors using bioinformatics analysis. METHODS: The pneumoconiosis and miRNA related reports were searched from the Google academic website. The miRNA sequencing or high-throughput microarray data sets based on the serum samplings of pneumoconiosis patients(case group) and normal healthy individuals(control group) were selected to screen for the differentially expressed miRNAs. Serum samples of patients with occupational silicosis and healthy controls were collected, and the relative expression of miRNAs was detected by real-time fluorescence quantitative polymerase chain reaction to verify the differential expression of miRNAs. The target genes of the differentially expressed miRNAs were predicted in the database of miRWalk, analyzed by Gene Ontology(GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway prediction. The transcription factor analysis of target genes was carried out by the database for annotation. RESULTS: Seven differentially expressed miRNAs were screened out and verified. Among them, five were up-regulated and two were down-regulated. GO enrichment analysis and KEGG signaling pathway prediction showed that the up-regulated differentially expressed miRNAs were mainly related to RNA polymerase Ⅱ promoter transcription and extracellular matrix, and were mainly involved in the occurrence and development of pulmonary fibrosis through adhesion plaque, protein digestion and absorption, phosphatidylinositol 3-kinase/protein kinase B and transforming growth factor-β signaling pathways. The down-regulated differentially expressed miRNAs were mainly related to the transcription of RNA polymerase Ⅱ promoter and the activity of DNA sequence specific transcription factors, that were mainly involved in the occurrence and development of pulmonary fibrosis through the signaling pathway of related hormone release. Transcription factor annotation results showed that SMAD family member 3, proto-oncogene JUN, forkhead box O1, early growth factor 1, β-catenin and other transcription factors may have an important relationship with the occurrence and development of pneumoconiosis. CONCLUSION: The seven miRNAs were differentially expressed in the serum of patients with pneumoconiosis. These miRNAs could be used as potential biomarkers for understanding the pathogenesis, the early diagnosis and treatment pneumoconiosis.

The Role of Fibrocyte in the Pathogenesis of Silicosis / 生物医学与环境科学（英文）

Exposure to free silica induces silicosis and myofibroblasts are regarded as primary effector cells. Fibrocytes can differentiate into myofibroblast. Therefore, the present study was designed to investigate whether fibrocytes participate in silicosis. The rat model of silicosis was established. Hematoxylin-eosin stainings and Masson stainings were used to evaluate the histopathology and collagen deposition. Flow cytometry and immunofluorescence were performed to detect the number of fibrocytes and their contribution to myofibroblasts. Results showed that fibrocytes participate in silicosis. Trend analysis of different sources of myofibroblasts during silicosis indicated that fibrocytes and lung type II epithelial cell-derived myofibroblasts play an important role in the early stage of silicosis, while resident lung fibroblast-derived myofibroblasts play a predominant role during the fibrosis formative period.

Crystalline Silica Promotes Rat Fibrocyte Differentiation in Vitro, and Fibrocytes Participate in Silicosis in Vivo / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The aim of this study was to investigate the effects of SiO2 on fibrocytes and whether fibrocytes participate in silicosis in vivo.</p><p><b>METHODS</b>A macrophagocyte (AM)/fibrocyte coculture system was established, and AMs were treated with 100 μg/mL SiO2. Flow cytometry was used to detect the number of fibrocytes. Real-time PCR was performed to measure the expression of collagen I, collagen III, and α-SMA mRNA. The levels of collagen I, collagen III, and TGF-β1 protein were determined by ELISA. Immunohistochemical staining was performed to measure α-SMA protein expression. A rat silicosis model was induced by intratracheal instillation of SiO2. Lung histopathological evaluation was conducted using HE and Masson's trichrome staining after 1 and 9 weeks. The number of fibrocytes in peripheral blood or lung tissue of rat was detected by flow cytometry. Double-color immunofluorescence was applied to identify fibrocytes in the lung tissue.</p><p><b>RESULTS</b>Peripheral blood monocytes were found to differentiate into fibrocytes in vitro in a time-dependent manner, and exposure to crystalline silica might potentiate fibrocyte differentiation. In addition, fibrocytes were able to migrate from peripheral blood to the lung tissue, and the number of fibrocytes was increased after SiO2 exposure.</p><p><b>CONCLUSION</b>Silica exposure potentiates fibrocyte differentiation, and fibrocytes may participate in silicosis in vivo.</p>

Establishing a genebank for phage single-chain antibody using silicotic rats / 中国职业医学

OBJECTIVE: To establish a genebank for phage single-chain antibody for further screening the specificity of single chain fragment variable( Sc Fv) in lung tissue of silicosis rats by phage display technology. METHODS: Twenty-four specific pathogen free male SD rats were used to construct silicosis model by one-time bronchial perfusion with 1. 0 m L of silicon dioxide suspension( mass concentration,100 g / L). We took periphery blood from 6 rats 3,6,9 and 12 weeks respectively after establishing the model. The peripheral lymphocytes were mixed,and total RNA was extracted using Trizol,and c DNA was synthesized by reverse transcription. The degenerated primers were used to amplify the variable region of heavy chain( VH) gene and variable region of light chain( VL) gene by polymerase chain reaction( PCR).Then VH and VL genes were assembled to form Sc Fv by T4 DNA linker. The cloning recombinant of Sc Fv and plasmid of PCANTAB-5e were transformed into competence E. coli TG1 by calcium chloride. The Sc Fv genebank of silicosis model was constructed by M13K07 helper phage superinfection. There were 10 bacterial colonies for plasmid restriction dualenzyme digestion randomly selected for confirmation. RESULTS: Agarose gel electrophoresis showed that there were two bands of obvious 28 S and 18 S in total RNA of periphery blood lymphocytes of silicosis rats. The total RNA was intact. The size of VH gene fragment was about 400 bp,the size of VL gene fragment was about 350 bp and recombinant Sc Fv gene fragment length was about 750 bp. The helper phage was amplified and placed with double-deck agar plate and observed limpid plaque with the size of a rice grain. The phage titer was 1. 35 × 10~(16) pfu / L. The recombinant plasmids were transformed into E. coli TG1 and total bacterial count was 8. 0 × 10~9 cfu / L in resistant plate. The positive cloned plasmid PCR gel electrophoresis and double enzyme results showed a positive inserting rate of 90. 0%. The capacity of phage single-chain antibody genebank of experimental silicosis was 7. 2 × 10~9 cfu / L. CONCLUSION: The silicosis rat model with phage Sc Fv gnebank could be successfully established,and its capacity and diversity provide support for the follow-up screening.

Protein expression in silica dust-induced transdifferentiated rats lung fibroblasts / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To analyze the expression of different proteins in free silica-induced transdifferentiated rat lung fibroblasts.</p><p><b>METHODS</b>Rat lung fibroblasts and alveolar macrophages were cultured. A transdifferentiation model of rat lung fibroblasts was established. Free silica was used as a stimulator for rat lung fibroblasts. Changes in α-SMA were detected by immunohistochemistry and Western blot, respectively. Protein of lung fibroblasts was extracted and analyzed by two-dimensional electrophoresis (2-DE).</p><p><b>RESULTS</b>Six protein spots were identified by mass spectrometry, including glyceraldehyde 3-phosphate-dehydrogenase, peroxiredoxin 5, heterogeneous nuclear ribonucleoprotein A2, transgelin 2, keratin K6 and vimentin.</p><p><b>CONCLUSION</b>Some proteins are changed in free silica-induced transdifferentiated rat lung fibroblasts.</p>

Role of insulin-like growth factor II receptor in transdifferentiation of free silica-induced primary rat lung fibroblasts / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the role of insulin-like growth factor II receptor in free silica-induced transdifferentiation of primary rat lung fibroblasts.</p><p><b>METHODS</b>Rat lung fibroblasts and rat alveolar macrophages were cultured. A transdifferentiation model of primary rat lung fibroblasts was induced by free silica. Levels of α-SMA protein, IGF-IIR protein and mRNA were measured by immunocytochemistry, Western blot and RT-PCR, respectively. Lung fibroblasts were treated with Wortmannin.</p><p><b>RESULTS</b>The expression levels of α-SMA and IGF-IIR increased with the increasing free silica concentration and decreased after Wortmannin was used.</p><p><b>CONCLUSION</b>The IGF-IIR plays an important role in free silica-induced transdifferentiation of primary rat lung fibroblasts.</p>

DNA methylation levels of genome in cFb transdifferentiation induced by SiO2 in rats / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the DNA methylation levels of genome in cFb transdifferentiation induced by SiO2 in rats.</p><p><b>METHODS</b>The primary macrophages and fibrocytes of SD rats were co-cultured directly and indirectly, which were exposed to SiO2 at the doses of 25, 50 and 100 g/ml. The transdifferentiation of cFb was identified with immunohistochemical assay. The genomic DNA methylation levels of cFb were detected with HPLC.</p><p><b>RESULTS</b>Under the condition of indirect co-culture, as compared with control group, the genomic DNA methylation levels of cFb exposed to SiO2 at the doses of 25, 50 and 100 g/ml reduced by 19.9%, 26.9% and 30.3%, respectively (P < 0.05); as compared with cFb exposed to 100 g/ml SiO2, the genomic DNA methylation levels of cFb exposed to 5-aza-dC decreased by 22.0% (P < 0.05). Under the condition of ThinCert(TM) direct co-culture, as compared with control group, the genomic DNA methylation levels of cFb exposed to SiO2 at the doses of 25, 50 and 100 g/ml reduced by 22.2%, 30.2% and 36.7%, respectively (P < 0.05); as compared with cFb exposed to 100 g/ml SiO2, the genomic DNA methylation levels of cFb exposed to 5-aza-dC decreased by 20.6% (P < 0.05).</p><p><b>CONCLUSION</b>Under the co-culture condition in vitro, SiO2 could reduce the genomic DNA methylation levels of cFb. The ThinCert(TM) direct co-culture can be used to study the silicosis fibrosis.</p>

A meta-analysis of susceptibility to pneumoconiosis and polymorphism of tumor necrosis factor-α 308 and 238 locus / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the relationship between the polymorphism of TNF-α gene 308, 238 locus and the susceptibility to pneumoconiosis.</p><p><b>METHODS</b>Eighteen published case-control studies about TNF-α gene 308, 238 locus polymorphism and pneumoconiosis susceptibility were searched out from sino-foreign databases from January 1994 to December 2010. Meta-analysis was applied on the published research to calculate the pooled OR value (95%CI) and stratified analyze the types and species of pneumoconiosis.</p><p><b>RESULTS</b>Eleven of the published research articles were selected into the analysis, including 10 research focusing on TNF-α gene 308 locus, with 1408 cases and 1639 controls in total. The meta-analysis showed that comparing with Gln/Gln carriers, Arg/Arg, Arg/Gln, Gln/Arg + Arg/Arg carriers were 1.89-fold (95%CI: 1.10 - 3.24), 1.53-fold (95%CI: 1.25 - 1.87), and 1.56-fold (95%CI: 1.28 - 1.90) more susceptible to pneumoconiosis, respectively. The stratified analysis showed that among coal workers, the TNF-α gene 308 locus Arg/Arg, Arg/Gln, Gln/Arg + Arg/Arg carriers were separately 2.29-fold (95%CI: 1.22 - 4.29), 1.56-fold (95%CI: 1.20 - 2.03), 1.64-fold (95%CI: 1.28 - 2.11) more susceptible to pneumoconiosis than Gln/Gln carriers; and among Asian people, the TNF-α gene 308 locus Gln/Arg, Gln/Arg + Arg/Arg carriers were separately 1.58-fold (95%CI: 1.28 - 1.95) and 1.57-fold (95%CI: 1.28 - 1.94) more susceptible to pneumoconiosis than the Gln/Gln carriers. Four case-control research focus on the study of TNF-α gene 238 locus, including 391 cases and 391 controls in total. The analysis showed that comparing with the non-carriers, TNF-α gene 238 locus Arg/Arg, Arg/Gln, Gln/Arg + Arg/Arg carriers were 6.03-fold (95%CI: 1.35 - 26.97), 1.87-fold (95%CI: 1.07 - 3.30) and 2.36-fold (95%CI: 1.37 - 4.07) more susceptible to pneumoconiosis.</p><p><b>CONCLUSION</b>TNF-α gene 308, 238 locus Arg/Arg, Gln/Arg, Gln/Arg + Arg/Arg carriers are more susceptible to pneumoconiosis.</p>