br according to the manufacturer s instructions All experi
> 20% according to the manufacturer's instructions. All experi-ments were performed in three replicates.
2.7. Gene expression profiling by RNA-sequencing
The whole transcriptome was sequenced in pair-end 150 bp using Illunima HiSeq X Ten. Reads were aligned to human reference genome, hg19/GRCh37. using STAR-2.5.3a (Dobin et al., 2013) with default settings. Annotated by ENSEMBLE release GRCh37, we calculated mRNA expression values as counts and FPKM using RSEM-1.2.30 (Li and Dewey, 2011). DESeq2 (Love et al., 2014) was applied to normalize the count data, calculate the fold change and do multiple testing corrections to measure the difference between BPS exposure and control sample. Significant genes were selected with the absolute fold change equal or more than 2 as well as p-value less than 0.05. GO and pathway enrichment were processed
2.8. Statistical analysis
Statistical analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, USA). Student's t-test was used to assess any possible statistical significance in DNA methylation level between control group and BPS exposure group. All p values were two-sided, and p values less than 0.05 were considered to be statistically significant.
3. Results and discussion
3.1. BPS exposure caused epigenetic changes in transposons of MCF-
In DNA methylation analysis, we focused primarily on trans-posons. Transposons are DNA sequences that can move from one location to another in the genome that comprise nearly half of the human genome (Lander et al., 2001). DNA methylation influences the transpose activity. When methylated, the transposon promoters are inactive and, over time, C to T transition mutations would occur at methylated sites, which could destroy many transposons (Yoder et al., 1997). Using a previously validated technique, which was referred as McrPCR, DNA methylation level changes in transposons were studied. In this method, a methylation sensitive endonuclease McrBC that requires two [A/G]mC half-sites to restrict DNA was used to digest genomic DNA in vitro. The targeted transposons were then amplified by PCR with specific primers according to a previ-ously report (Rabinowicz et al., 2003). The difference in amount of PCR products in DNA samples with and without McrBC Pam3CSK4 might indicate the methylation level of the sample.
Based on previous studies about the epigenetic effects of BPA (Mlynarcikova et al., 2013; Bhan et al., 2014; S¸ enyildiz and Ozden, 2015), in present study, we selected 10 nM, 100 nM and 1 mM exposure concentrations of BPS for 24 h for the analysis of changes in DNA methylation. In total, 8 tansposons from randomly selected genes were analyzed, including Ac-like, HERVE, LINE 2, MaLR, Mariner 2, MER 2, MER 4 and SINE-R (Table 1). As shown in Fig. 1, by comparing the sample digested and undigested (take lane 1 and lane 5 as an example), we found that after digestion, the amount of amplified transposons were decreased, indicating that they were methylated. When the same amount of DNA from the control and BPS exposure group were subjected for McrBC digestion (lane 1 to 4), compared with the 5th lane, the weaker or stronger lane of the BPS exposure group (lane 6 to lane 8) means that BPS exposure
Fig. 1. Methylation level changes in transposons of MCF-7 cells under exposure of BPS.
changes methylation levels of the transposons. Our results showed that for MaLR and Mariner 2, the amplified transposons in the exposure group with 10 nM and 100 nM BPS were the same as control group, while that in the 1 mM BPS group were decreased, indicated that 1 mM of BPS exposure induced the hypermethylation of these transposons. While for MER2, 100 nM BPS can also induce its hypermethylaton (Fig. 1). The results suggested that BPS expo-sure could increase the methylation level of transposons.
It was reported that BPA could cause epigenetic changes, which was one of the possible mechanism through which BPA induces human diseases (Rezg et al., 2014). However, there was no formal assessment on whether BPS could induce epigenetic effects on human. Our results here showed that BPS exposure influenced the methylation status of transposons. Transposons are firmly regu-lated from early embryonic development and during the entire course of human life. A growing number of studies have delineated that epigenetic mechanisms may also control transposon reac-tivation with subsequent effects on carcinogenesis (Chenais, 2015). DNA methylation in transposons is a key mechanism defending against their transposition activities (de la Rica et al., 2016; Hu et al., 2017; Zheng et al., 2017). As DNA methylation status affects the mobility of transposons and then influences the genomic instability as well as induces oncogenic activation and transcriptional dysre-gulation, in this work we analyzed changes of DNA methylation in transposons induced by BPS to evaluate the effects of BPS on breast cancer cells. We found that BPS exposure increased DNA methyl-ation levels of several of the analyzed transposons. This result not only indicated that BPS could induce genomic DNA methylation changes, but also implicated that BPS may contribute to dysregu-lation of gene expression.