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r> However, to the best of our knowledge, information on the epigenetic effects of BPS is lack at present. While it is too early to speculate whether the epigenetic effects of BPS is similar with BPA, the similarity in their structure and common endocrine-disruption effect warrants investigation on the potential of BPS to induce epigenetic and related transcriptional changes in organisms. In this work, we aimed to investigate whether BPS could also cause epigenetic and transcriptional changes in human breast cancer cell 
2. Materials and methods
Bisphenol S (BPS), dimethylsulfoxide (DMSO) were bought from Sigma-Aldrich (St. Louis, MO, USA). Human breast cancer cell line MCF-7 (ATCC NO. HTB-22) was purchased from the American Type Culture Collection (ATCC). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), streptomycin sulfate and peni-cillin, 0.25% trypsin were obtained from Gibco (ThermoFisher Sci-entific, USA). 10 cm and 6 cm cell culture dishes were obtained from Corning (New York, USA). McrBC enzyme was from New England Biolabs Inc. (NEB, Ipswich, MA, England). SYBR Premix Ex Taq™ (Tli RNaseH Plus) was from Takara Biotechnology (Mountain View, CA, USA).
2.2. Cell culture and treatment
MCF-7 cells were cultured under the recommended conditions. Briefly, MCF-7 cells were maintained in DMEM supplied with FBS to a final concentration of 10% and streptomycin sulfate and penicillin to final concentrations of 100 units/mL and 100 mg/mL under an RGDS peptide of 5% CO2 at 37 C. BPS was dissolved in DMSO to get a stock solution of 10 mM and then serially diluted to get the final concentration of 1 mM, 100 nM and 10 nM. Following a 48-h incu-bation to allow cell attachment, medium was changed to include various concentrations of BPS. Medium contains the same amount of DMSO was used as control group. After the addition, culture of the cells was continued for another 24 h before harvest.
2.3. Genomic DNA extraction
Genomic DNA of the treated cells was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, Wis-consin, USA) according to the manufacturer's recommendation. Briefly, cells were lysed with the nuclei lysis buffer, then RNA was eliminated by RNase digestion followed by protein precipitation. Genomic DNA was finally precipitated by isopropyl alcohol. The concentration and purity of the DNA were detected by NanoDrop spectrophotometer (ThermoFisher Scientific, USA).
2.4. Total RNA extraction
Total RNA was extracted from the treated cells by TRIzol reagent (Invitrogen, ThermoFisher Scientific, USA) according to the manu-facturer's recommendation. The concentration and purity of the RNA samples were detected by NanoDrop spectrophotometer.
2.5. Analysis of changes in DNA methylation levels by McrPCR
A previously reported PCR-based technique named McrPCR was employed to analyze the changes in DNA methylation levels caused by BPS exposure in specific DNA sequences (Doerks et al., 2002). McrBC digestion was performed as recommended by the supplier (NEB). Briefly, 33 mL genomic DNA (500 ng) was firstly digested by 2.0 mL McrBC (20 U) in the presence of 0.5 mL GTP (2.0 mM), 0.5 mL BSA (100 mg/mL) and 4.0 mL NEBuffer at 37 C overnight. The digestion was stopped by incubation at 65 C for 20 min.
Table 1
Primer sequence of the targeted transposons in McrPCR analysis.
Transposons Accession number Primers Size (bp) CpG sites
Ac-like Y17156 Left-CACGCTCAACATCAAGGAGA 271 17
Right-CGGGAAGATCTTGTCCTCAG
HERVE AF026246 Left-ACAACCCTTGCAGAGTGACC 216 5
Right-TTGCTGTCCTACTTCGGTGA
LINE 2 AC002299 Left-AGACCCTCCAATGACTTTCC 478 5
Right-AGGCTCTGAAATCACAGTGG
MaLR AC105289 Left-ATCACAAACAAGGCGGTTTA 426 3
Right-TTAGAATGGATGGACCCAAA
Mariner 2 AP001706 Left-CAAAGAGTTCATGGAAAGTGC 421 3
Right-CCATCAATTAGAACACGAACC
Right-ATCCTTGAACAACGTGCATT
Right-ATTGATAACTGGGGTGAGCA
SINE-R AB062085 Left-GGATTAAGGGTGGTGCAAGA 209 3
Left-TGGCAGGGTCACAGGATAAT
72 C. An extension of 10 min at 72 C followed the last cycle. The PCR products were analyzed on 2.5% agarose gel stained with GelRed in 1 TAE buffer under the voltage of 300 V for 20 min.
2.6. PCR array analysis of methylation status in gene promoter
The analysis of methylation level in promoter of specific genes was conducted using the custom EpiTect Methyl II PCR system (Qiagen, Chatsworth, CA), which is based on the detection of remaining input DNA after restriction digestion with methylation sensitive and methylation dependent enzymes. The restriction di-gestions were performed using the EpiTect Methyl II DNA Restric-tion Kit according to the manufacturer's recommendation. In brief, a 120 mL reaction mix containing 1 mg genomic DNA, 26 mL of 5 Restriction Digestion Buffer, and nuclease-free water was firstly prepared. Then four digestion reactions (Mo, Ms, Md and Msd) were set up, which consist of 28 mL previous reaction mix and 2 mL nuclease-free water for the Mo digest, 1 mL methylation sensitive enzyme A and 1 mL nuclease-free water for the Ms digest, 1 mL methylation dependent enzyme B and 1 mL nuclease-free water for the Md digest, 1 mL methylation sensitive enzyme A and 1 mL methylation dependent enzyme B for Msd digest. All four digestions were conducted at 37 C overnight. Then the digested samples were analyzed with the Human Breast Cancer EpiTect Methyl II Signature PCR Array according to the manufacturer's recommendation. Methylation status of 22 breast cancer-related genes was analyzed. The Real-time PCR assay was performed in a StepOnePlus™ real-time cycler (Applied Biosystems, Foster City, CA). Data were analyzed using the provided integrated Excel-based template. Methylation level change was considered to be hypermethylated if