The Effect Of Silica And Carbon Black On MAPK/AP-1Cell Signaling Pathway

Silicosis and carbon black (CB) pneumoconiosis are legal occupational disease in China induced by silica and CB. Inflammatory reaction plays an important role in the process of the occurrence of pneumoconiosis, and MAPK/AP-1cell signal transduction pathway is also involved. In recent years, many studies have found that gene expression not only depends on gene itself, but also on epigenetic modifications which are defined as no changes in gene sequence. There is no report about the role of genome-DNA methylation in silicosis patients, DNA methylation regulation of MAPK/AP-1signaling pathway and the role of this pathway in HELF induced by CB.Our study was divided into two parts, research materials were ten Formalin-Fixed Paraffin-Embed (FFPE) tissue of silicosis patients lung tissue and two normal lung tissue, and silicosis groups were divided into early group (6cases of I phase silicosis) and advanced group (2cases of II phase silicosis and2cases of III phase silicosis) and all the cases were excluded lung cancer, and Human Embryonic Lung Fibroblasts (HELF) in the first part. Infinium450K was used to investigate the genome-scale DNA methylation change from FFPE and the protein level of PTEN and c-Jun were determined by immunohistochemical in FFPE. The methylation of PTEN and c-Jun gene were analyzed by MS-PCR in HELF. The results were as follows:1) Compared with the normal lung methylation data from GEO database, silicosis FFPE samples were showed abnormal genome-scale methylation change, of which86770CpG sites and79660CpG sites significantly differed in methylation status in early-stage and advanced stage. Among all significantly CpG sites in these two groups,7.3%and17.4%were significantly hypermethylated, and about60%were associated with gene promoter in both groups, and92.7%and82.6%were the low methylation sites.2) The change of sites associated with PTEN and c-Jun gene in Infinium450K. Infinium450K covered63loci,25loci and19loci associated with PTEN, Jun and Fos, of which53,19and1was related with the promoter region, respectively. According to the criteria of P≤0.001and difiference score|≥0.4to analyze the sites with promoter region, there was4hyper-methylated loci associated with PTEN in early group, and10hyper-methylated loci associated with PTEN and3hyper-methylated loci associated with Fos in advanced group. There were no hypo-methylated sites associated PTEN, Jun and Fos.3) KEGG analysis revealed many signaling pathway were considered significant, even the same cell signaling pathway showed different sensitivity in these two groups.4) H&E staining results showed lung biopsy contained lots of inflammatory cells infiltration around the bronchi, blood vessels and its lumen in the early group, but in the advanced group fibroblasts, collagan fibers and dust cell further proliferation formed typical silicon nodules. Early-stage group showed the positive expression of c-Jun and PTEN protein and negative or mild expression in advanced-stage group.5) PTEN showed unmethylated and methylated band at all time points and c-Jun showed methylated stripe in3h and6h and methylation band dispeared in12h in HELF detected by MS-PCR.The second part of this study analyzed the effect of silica and CB on CTGF and the role of MAPK on cytokines induced by CB by using Werstern Blot, Dominant Negative Mutant, Electron Microscope Scanning, Luciferase Detection and Immunofluorescence Technique. MTT results showed that CB concentration between60to240μg/ml, it was not obvious that the cell survival rate declined and the corresponding cell survival rate was65%-80%, so the subsequent experiment chose100μg/ml as treated dose. CB particals were not observed but silica partical were in HELF cells by Electron Microscopy. siRNA-CTGF were transfected into HELFs which was selected by G418and indentified by Fluorescence Microscope and Werstern Blot. CTGF level was increased after exposure to100μg/ml silica12h and100μg/ml CB24h. CB caused IL-6and IL-8change by MAPK cell signaling pathway and not dependent on AP-1. Compared with our previous study, silica could cause abnormal cell cycle change and CB could cause cytockines change by MAPK, all of which indicated MAPK played an important role in particals exposure.