The Regulation Of MiR-18a By P53in The Arsenic Induce Re-expression Of Estrogen Receptor α

Breast cancer is the most common tumor in women, and it is the major cause of morbidity and mortality. The expression of estrogen receptors (ERs) is a marker for prognosis and is predictive response to endocrine therapy. About two-thirds of breast cancers express the ER gene and synthesize the ERa protein. These tumors tend to be more differentiated and are often responsive to hormonal therapies. One-third of breast cancers loses the ERa, Estrogen receptor (ER)-negative breast carcinomas are often difficult to treat as they do not respond to hormone therapy. In addition, some breast cancers that are initially ER-positive lose ERa expression during tumor progression. Thus, new strategies are needed for re-expression of ERa and thereby for sensitization of ER-negative breast cancer cells to selective ER modulators. To date, the molecular mechanisms underlying the loss of ERa in breast cancer are poorly understood. Loss of ERa is related with some microRNA expression levels. Recently, the gene ESR1, which encodes the estrogen receptor-a (ERa), was identified as a target of many microRNAs. microRNAs can repress ERa translation by binding to its mRNA at the3’-untranslated.region. In addition, studies demonstrated that p53upregulates estrogen receptor-a (ERa) expression in the human breast cancer cell line. We have found that arsenic induces functional re-expression of ERa by demethylation of DNA in ER-negative breast cancer. However, the level of ERa expression by arsenic trioxide in MDA-MB-231cells is lower than in MCF-7cells, which ER-positive, indicating that there could be other mechanisms involved in silencing of ERa. Whether arsenic trioxide can re-express ERa in ER-negative breast cancer cells by regulate p53or miR-18a expression have not been reported.Objectives:In the present study, the ER-negative breast cancer cells (MDA-MB-231and Hs578T cells) were used as in vitro models, and the MDA-MB-231female nude mice xenograft model was used as a in vivo model. To investigate the mechanism of re-expression of ERa induced by arsenic trioxide in ER-negative breast cancer cells, and provide information regarding the potential implication of arsenic trioxide as a new drug for treatment of ER-negative human breast cancer.Methods:The expression of ERa mRNA and protein induced by arsenic trioxide in MDA-MB-231cells, Hs578T cells and nude mice xenograft model were observed with RT-PCR and Western blotting. The function of re-expression of ERa detected by MTS assay which detected cell proliferation, RT-PCR which detected the expression of ERa target genes. The expression of wtp53induced by arsenic trioxide was detected by Western blotting assay; the expression of miR-18a was detected by real-time PCR.Results:A certain concentrations of arsenic trioxide induced re-expression of ERa mRNA and protein in ER-negative human breast cancer cells. The re-expression of ERa induced by arsenic trioxide was associated with increased basal levels of expression of the ER-responsive genes, pS2and GREB1in the presence of E2. The quantitative PCR results showed that in ER-negative breast cancer MDA-MB-231cells, arsenic trioxide down-regulated the expression of miR-18a, miR-19a, miR-19b. The expression of ERa in MDA-MB-231cells was increased by silencing miR-18a. In contrast, the expression of ERa and ER-responsive genes, pS2and GREB1were down-regulated by ectopic expression of miR-18a in MCF-7cells. Arsenic trioxide-induced the expression levels of ERa, ER-responsive genes, pS2and GREB1were blocked by miR-18a-mimics in MDA-MB-231cells. In addition, the degree of growth stimulation/inhibition by exposure to E2, OHT or ICI restored by arsenic trioxide was also reversed by miR-18a-mimics in MDA-MB-231cells. These results suggest that re-expression of functional ERa induced by arsenic trioxide is mediated by miR-18a. Furthermore, we investigated the possible upstream regulator of miR-18a. Western blotting assays showed that arsenic trioxide induced the expression of wild-type p53expression and phosphorylation of p53. When the cells were transfected with wild-type p53plasmid or added wild-type p53inducer (CP-31398), the expression of miR-18a was reduced. The expression of miR-18a can be regulated by wild-type p53. Meanwhile, transfected with wild-type p53plasmid could increase the expression of ERa; we performed transfection analysis using wild-type p53and mutated p53plasmid after MDA-MB-231cells treatment with2μmol/L arsenic trioxide for6days. Overexpression of wild-type p53significantly inhibited the miR-18a, miR-17a, miR-19a and miR-19b expression and increased the ERa levels induced by the arsenic trioxide, in contrast overexpression of mutated p53significantly increased the miR-18a, miR-17a and miR-19a expression and inhibited the ER levels induced by the arsenic trioxide.Conclusions:A certain concentrations of arsenic trioxide induced fuctional re-expression of ERa mRNA and protein in vitro and in vivo, wild-type p53-mediated miR-18a plays a critical role in arsenic trioxide-induced re-expression of ERa.