| ObjectivePneumoconiosis accounts for most of the occupational disease in China, has been affecting the living quality of pneumoconiosis patients and the economic development of the society. The silicosis is the the most severe disease caused by free Si O2 in Pneumoconiosis. The mechanism of development of silicosis is not known. In a recent study showed that DNA methylation was the possible regulative way to the transdifferentiation of fibroblast in silicosis. In the past research, the level of genome methylation was examined by HPLC showed that the variation of DNA methylation was related to the silicosis. However, the detection of specific genes was absenced. In this study, we examined the DNA methylation used Me DIP-seq to find different DNA gene. Our aim was looking for the possible intervention targets and provided the new evidence to the treat of silicosis. MethodThe pulmonary fibroblasts were extracted from the SD male rat by tissue explant and cultivated to 6-8 generation. The pulmonary macrophages were extracted from the SD male rat by bronchoalveolar lavage. Co-culture model were build used the transwell. Infected macrophages using 100 ug/ml of Si O2 solution, and collected the fibroblasts respectively in 0 h(A1)(A2), 24 h, 48 h(A3). Extracted DNA from the fibroblasts used the QIAamp DNA Mini Kit. Briefly, DNA methylation were examined used Me DIP-seq. The raw data were disposed by removing the low quality data and used for the subsequent analysis. The distribution characteristics in the component of gene of reads and peaks were described. Moreover, differential methylated regions and genes were analyzed. The bioinformatics of differential genes in the three groups were analyzed used the DAVID. The validate of the data of the Me DIP-seq were used bisulfite sequencing. ResultMe DIP-seq data shoewed that 26,048,428(60.90%)、34,162,578(61.53%)、33,314,252(60.18%)reads were mapped to reference genome, and this reads were used for the following analysis. 208,253 peaks, 206,685 peaks and 232,504 peaks were found in the three groups respectively and 208,253 peaks, 206,685 peaks, 232,504 peaks were found mapped to the component of gene. The analysis of read and peak showed that the intergenic enriched the most number of the reads and peak followed by intron. A further analysis of differentially methylated regions and genes showed that 8791 different DNA methylated genes were discovered in all the three groups, analysis about those genes in GO and KEGG pathway were carried out used DAVID subsequently. The analysis of GO showed that different DNA methylated genes were enrichmented in one or more of the following three categories: biological process(n=4402), cellular component(n=3920), and molecular function(n=4290). Transitorily, the analysis of KEGG pathway about the different DNA methylated genes were found enrichmented in metabolism, environmental information processing, cellular processes, organismal systems, human diseases. And those pathways were subdivided to 37 pathways. ConclusionThe fragments of the DNA methylation in the transdifferentiation were most enriched in the intergenic region followed by exon and intron. Just a few of the fragments were found in the promoter and CPG islands.A large number of DNA methylated genes were found. The function of the genes was contacted with phosphorus metabolism, plasma membrane and ion channel.The transdifferentiation of the fibroblast may be contacted with the energy metabolism of mitochondria. Adhesion plaque was the main component after the transdifferentiation. Some methylated genes were found contacted with the cynapse. The process of transdifferentiation was similar to the development of cancer and MAPK, Wnt, Erb B, p53 and TGF-β pathway played an important role in this process possibly. |
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