br Mechanism by which BPS induced DNA methylation may simila
Mechanism by which BPS induced DNA methylation may similar to other environmental chemicals. The mechanism of environ-mental chemicals induced changes in global DNA methylation status may rely on two often described hypotheses. First, evidence has demonstrated the direct action of environmental chemicals on the function of DNMT and TET enzyme families. Second, evidence has also shown that environmental chemicals may change the availability of SAM. Besides, changes in gene-specific DNA methylation patterns may because of that environmental chemicals exposure trigger transcription factors, and the presence or absence of transcription factors on the DNA denies or allows access to the DNA methylation, then influence the observed site-specific pat-terns of methylation (Martin and Fry, 2018).
3.2. BPS exposure changed methylation level of breast cancer-related genes
The Human Breast Cancer EpiTect Methyl II Signature PCR Array was used to analyze the changes in methylation status of 22 tumor
suppressor gene promoters whose hypermethylation was reported to occur frequently in a variety of breast tumors. The results showed that the methylation status of BRCA1, CCNA1, CCND2, CDH1, CDH13, CDKN1C, MGMT, PRDM2, PTEN, PTGS2, PYCARD, SFN, THBS1, TNFRSF10C, ESR1 in the exposure group of 1 mM BPS differed significantly (p < 0.05) from the methylation status of control group. Most of these genes showed higher methylation, except CCND2, CDH13 (Table 2). However, according to the instructions of the manufacturer, a threshold of 20% changes in methylation level was considered to be hypermethylated or hypomethylated. Only methylation level changes of CDH1, SFN, TNFRSF10C exceeded the threshold for hypermethylation (Table 2).
Abnormal methylation of the CpG islands in the promoter re-gions is related to gene inactivation. It has been described as a mechanism in many cancer types. Particularly, aberrant methyl-ation of the promotor region of a gene could lead to gene silencing, which would contribute to the initiation or malignant progression of tumors (Klutstein et al., 2016). In normal cells, CpG islands are generally hypomethylated or unmethylated and maintain a tran-scriptionally favorable local L-Glutamine configuration. Thus, the normal expression of these genes would inhibit the occurrence of tumor. However, in cancer cells, the CpG islands in these regions are hypermethylated, which leads to alterations in chromatin confor-mation, and then the tumor suppressor genes will be silenced. As a result, cells will enter cell cycle that is accompanied by loss of apoptosis, defects in DNA repair, angiogenesis, and loss of cell adhesion. These biological processes would eventually lead to tumorigenesis (McCabe et al., 2009). As the methylation status in CpG islands is dynamic, the changes in methylation level is in accordance with the degree of malignancy of the tumor.
CDH1 is a key gene that relates to the activity of cell invasion. The loss of CDH1 function would benefit tumor cells from acquiring the invasive properties of metastatic tumor cells (Graff et al., 2000). SFN gene encodes stratifin, the loss of which was suggested to contribute to malignant transformation of tumors through impairing the function of G2 cell-cycle checkpoint, inducing an accumulation of cellular genetic defects (Hermeking et al., 1997). The hypermethylation of CpG islands in SFN gene has been detected
Comparison of methylation level of the analyzed gene promoters in control group and BPS exposure group.
Genes Methylation level (%)
in 91% of breast tumors (Ferguson et al., 2000). The similar phe-nomenon was reported for the TNFRSF10C gene as hyper-methylation of its promoter CpG island (CGI) had been reported in many tumors, including primary breast cancer (33%) (Cheng et al., 2009). The increased methylation level in promoter of these genes suggested that BPS exposure might participate in promoting the degree of malignancy of breast cancer.
3.3. BPS exposure changed gene expression levels
Gene expression profiles of the control and BPS exposure groups were generated. Fig. 2 showed the heat map resulted from the hi-erarchical clustering method and the principal component (PC) plot. The PC analysis (Fig. 2A) demonstrates the relatedness of the samples. Visually, the BPS exposure group segregated apart from the control group. This result is corroborated by the heat map representation of the gene expression levels (Fig. 2B). The obtained results suggested that BPS exposure induced a strong effect in gene expression of breast cancer cells. In total, expression of 156 genes were significantly changed by BPS exposure (Table S2). We noticed that several genes related to development of breast cancer were up-regulated, including THBS4, PPARGC1A, CREB5, COL5A3. THBS4 is related to the cellular attachment, adhesion and migration, as well as proliferation. The expression of THBS4 in breast cancer-associated extracellular matrix would help to activate stromal