| Objective:Build Yunnan Province Xuanwei bituminous coal combustion to release substances induced malignant transformation of bronchial epithelial cells cell model; through the genome methylation chip combining transcriptome microarray and analysis of soot pollution lead to apparent correlation between genetic alterations are associated with lung cancer and found a group of soot pollution related lung cancer early molecular markers and for the local high incidence of lung cancer of early prevention, early diagnosis, early treatment provide valuable scientific data.Methods:1.Collecting coal buring emission made of solution,and using the 1.0 ug/ml CBE induced liquid on human Bronchial epithelial cells (BEAS-2B) B2b in vitro to establish the malignant cell model C-B2b (Coal-Beas-2b);2. Observeing the biological characteristics of C-B2b cells;3.Whole MeDIP-Chip and Microarray analyses detected the MUC1 gene methylation and mRNA expression level of difference in B2b, C-B2b and Xuanwei lung cancer cell (XWLC-05);4.Making GO(ontology gene) classification to explore its biological function for differential expression of mRNAs;5.KEGG (Encyclopedia of Genes Kyoto and Genomes) signaling pathway analysis and differentially expressed genes related signaling pathways;6.1t made use of qRT-PCR to verify the level of mRNA expression of MUC1 gene;7.Data statistics and analysis using SPSS 17.0 software package, the data to mean+standard deviation (s), the group of data between the application of single factor analysis of variance (WAY ANOVA ONE) and t test.Results:1.It established the malignant transformation of C-B2b cell lines by induction culture in vitro;2.Compared the malignant transformation of C-B2b cells to B2b, it had obvious biological characteristics;3.Compared with Beas-2b, MeDIP-Chip array found 1872 promoters hypermethylated as well as 3182 promoters were demethylated in C-B2b, and 1720 promoters were hypermethylated as well as 4037 promoters were demethylated in XWLC-05 cells. Among the promoters,595 hypermethylated promoters as well as 2164 demethylated promoters were shared both in C-B2b and XWLC-05 cells;4.Through analysis the result of, we found that C-B2b cells to 2465 transcripts were up-regulated, down 2574 transcripts; XWLC-05 cells is 3959 transcripts were up-regulated,3763 transcripts down regulated. In contrast to C-B2b and XWLC-05, the same 1257 transcripts were up-regulated and 1431 transcripts were down regulated;5. GO analyses categoried differentially expressed mRNAs as three GO families: biological process, cellular component and molecular function. And showed a variety of biological functions of differentially expressed mRNAs;6.KEGG pathway results showed that 33 pathways were up-regulated and 16 pathways were down-regulated in C-B2b, and 24 pathways were up-regulated and 53 pathways were down-regulated in XWLC-05. And the most Enriched up-regulated pathways in XWLC-05 cells, including Influenza A, Cytokine-cytokine receptor interaction and NF-kappa B signaling pathways, were also up-regulated in C-B2b;7.qRT-PCR to verify the expression of MUC1 in MeDIP-Chip.Conclusion:1.The CBE will lead to malignant transformation of B2b cells;2.Establishing the C-B2b model of malignant transformation cells successfully,which lay the foundation for further research of Xuanwei lung cancer;3.MeDIP-Chip and Microarray analyses confirmed that, B2b as the reference cells, C-B2b cells and XWLC-05 have similar methylation profile and gene expression profile. A number of possible bioaccumulation process and pathway enrichment provided by comprehensive information, which provides the direction and basis for the follow-up study of lung cancer correalated with coal pollution.4.MUC1 gene related mRNA differential expression profile provides a number of possible enrichment biological processes and enrichment pathways, which provide the direction and basis for the subsequent research;5.The results of qRT-PCR were consistent well with the microarray results, suggesting that the results is reliable. |
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