| Chlorophenols (CPs) as the persistent organic pollutants have been widely used for medicine, agricultural and manufacturing production. CPs caused inevitably environmental pollution. CPs can exist stably in the environment especially in the water. CPs do not only affect the growth of various aquatic organisms, but also can endanger human health.2,4-dichlorophenol (2,4-DCP), one of the most abundance of CPs in the environment, is used as solvents, pesticides and pharmaceutical intermediates, and has been found to be harmful to humans and animals. Therefore, 2,4-DCP has been defined as priority pollutants in USA, China and many other countries.The transcriptome sequencing technology based on Illumina/Solexa sequencing platforms is capable of detecting single nucleotide at the overall transcripts level in any species, and analyzing the structure of transcript and the gene expression levels. Meanwhile, this technology could also detect the unknown or rare transcripts and provide the most comprehensive information. Compared with the conventional DNA microarray, this technology could be performed with neither pre-designed probes for known sequences nor reference sequences. In a word, transcriptome studies could reveal the gene structure and gene function on the overall level, and clarify the specific biological processes and the molecular mechanisms of some diseases as well.The existing studies on 2,4-DCP mostly concentrated on its environmental degradation and the studies that to clarify the toxic mechanisms almost focused on the determination of multiple physiological indicators. However, the studies on 2,4-DCP toxicity combined with transcriptome sequencing technology has not been reported yet. In the present study, the transcriptome data were processed to do sequence alignment, functional annotation and metabolic pathway analysis through the establishment of transcriptome database in 2,4-DCP-treated HL-7702 cells. Further more, according to the analysis of differentially expressed genes under 2,4-DCP exposure, the genes related to 2,4-DCP toxicity were screened and used to do further investigation on the potential mechanisms of 2,4-DCP toxicity. The results of this study are as follows:1. In this study, the raw data obtained by RNA-seq were about 7.77 GB, in which the control group and 2,4-DCP-treated group got 54,711,542 and 51,353,236 high quality pair of short sequences respectively.In these short sequences, the total reads which could be compared to the reference sequence were 45,318,071 in the control group and 42,090,330 in the 2,4-DCP-treated group. Moreover, the genes covered 90% to 100% of the total number of genes were 56% and 55% in the control group and 2,4-DCP-treated group respectively.2. After the analysis of differentially expressed genes in control group and 2,4-DCP-treated group,1521 differentially expressed genes (FDR≤0.001 AND |log2Ratio|≥1) were screened, in which 795 genes were up-regulated and 726 genes were down-regulated.3. By GO and KEGG analysis, these differentially expressed genes turned out to be mainly involved in steroids synthesis, cell metabolism, tissue development, endoplasmic reticulum stress and other biological processes.4. RT-qPCR assay was used to identify the expression level of 10 genes (ATP8A1, ATP8B4, CYP1A1, CYP1B1, CYP2E1, CYP4F12, UGT1A6, UGT1A7, DHCR7 and DHCR24) that affected significantly by 2,4-DCP, and the results were accordant with those by transcriptome analysis. The expression of these genes changed a lot in HL-7702 cells treated by 2,4-DCP since they are involved in protecting the cell membrane (ATP8A1 and ATP8B4), mediating the metabolism of 2,4-DCP (CYP1A1, CYP1B1, CYP2E1, CYP4F12, UGT1A6 and UGT1A7) or regulating the synthesis of intracellular steroid substances (DHCR7 and DHCR24). These results also demonstrated that the transcriptome analysis could reflect the true expression of genes in cells.Through the study mentioned above, we found that at least 3 different toxic mechanisms involed in HL-7702 cells treated by of 2,4-DCP were listed as the following:(1) 2,4-DCP affect the stability of cell membrane structure by changing phospholipids structure; (2) 2,4-DCP alter the normal physiological function of cells by producing more toxic metabolic products or intermediate substrates; (3) 2,4-DCP play a role for endocrine disruption by changing the synthesis of intracellular steroid substances.In conclusion, our study represented the most comprehensive characterization by RNA-seq transcriptome profile including differential expression genes, gene ontology analysis and pathway analysis in 2,4-DCP-treated HL-7702 cell, which provided important clues for understanding the molecular mechanisms of 2,4-DCP toxicity at system-wide levels. |
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