Change Of Epigenetics In Malignant Transformation Of BEAS-2B Cells Induced By Coal Tar Pitch Extracts

ObjectiveEpigenetic including DNA methylation, histone modifications and non-codingRNA regulation, which can regulate the expression level and function of gene whiledoes not involve DNA sequence changes. And epigenetic abnormalities often occurearly in the process of carcinoma. Therefore, in this study, coal tar pitch extract(CTPE) was used as a test object to induce the malignant transformation model viahuman bronchial epithelial (BEAS-2B) cells in vitro. After being confirmedmalignant transformation of BEAS-2B cells, the genomic DNA methylation levels,the mRNA and protein expressions of DNA methyltransferase1(DNMT1), DNAmethyltransferase3a (DNMT3a), DNA methyltransferase3b (DNMT3b), histonedeacetylase enzyme1(HDAC1) also as miR-21and miRlet-7a expression levels weredetermined. The malignant transformation mechanism induced by CTPE wasexplored especially in the early stage of precancerous lesions. The expressionchanges of epigenetic molecular markers may provide more efficient signs for thediagnosis of lung cancer and estimating risk in early stage.MethodsThree groups were divided as dimethyl sulfoxide group (DMSO),benzo(a)pyrenegroup (B(a)P), the extracts of coal tar pitch group (CTPE). According topre-experiment exposure dose, the malignant transformation model was establishedvia BEAS-2B cells. The genomic DNA methylation level was measured by highperformance liquid chromatography (HPLC). Real-Time PCR was used to detect therelative gene expression levels of DNMT1, DNMT3a, DNMT3b, HDAC1mRNA,miR-21and miR let-7a. The protein expressions of DNMT1, DNMT3a, DNMT3band HADC1were measured by enzyme-linked immunosorbent assay (ELISA).Results1. The genomic DNA methylation level: At the passage of10th, there were nosignificant differences among three group in the genomic DNA methylation rate (P=0.069). At the passage of20th, the genomic DNA methylation rates in B(a)Pgroup and CTPE group were significantly decreased than that in DMSO group(P<0.001; P<0.001). The rate in B(a)P group was lower than in CTPE group andthere was statistically significant difference (P=0.042). At the passage of30th, themethylation rates significantly decreased among all three groups (P=0.003), whilethere was no significant difference between B(a)P group and CTPE group (P=0.322).2. DNMTs' mRNA and protein expressions: There were no significantdifferences in DMSO group with increasing passage in the mRNA and proteinexpressions of DNMT1, DNMT3a, DNMT3b (all P>0.05). The mRNA and proteinlevels of DNMT1were significantly increased between B(a)P group and CTPE group(all P<0.05). The mRNA and protein levels of DNMT3a were significantlyincreased between B(a)P group and CTPE group(all P<0.05). While the mRNA andprotein levels of DNMT3b were significantly increased in B(a)P group (P=0.005,P<0.001). There was no significant difference in the mRNA expression of DNMT3bin CTPE group (P=0.131) while significant difference for the protein (P<0.001).3. HDAC1and protein expressions: With the passage increases, the levels ofmRNA and protein of HDAC1were significantly increased between B(a)P group andCTPE group (all P<0.05). There were no significant difference in DMSO group(P=0.997; P=0.326).4. miR let-7a and miR-21relative gene expression levels: Similarly, there weresignificantly increased in the relative expression levels of miRlet-7a between B(a)Pgroup and CTPE group(P=0.013; P=0.002). There were no significant difference inDMSO group (P=0.996).There were significantly increased in the relative geneexpression of miR-21between B(a)P group and CTPE group as passagesincreased(P=0.012; P=0.034). While there were no significant difference in DMSOGroup (P=0.999).Conclusion1. CTPE carcinogenicity related with low genomic DNA methylation, theexpression levels of DNMTs, HDAC1, miR-21, miRlet-7a significantly increased andthese factors may co-regulate gene expressions.2. These epigenetic molecular markers changed in malignant transformation ofBEAS-2B cells could be used to be efficient signs for the diagnosis of lung cancer andestimating risk in early stage.