| Histone methyltransferase SMYD3 (SET and MYND domain-containing protein 3) is an important regulatory factor in breast cancer. In addition, the abnormal activity of estrogen signaling pathway and estrogen metabolites are also closely related with the occurrence and development of breast cancer. However, so far, the effect of SMYD3 on estrogen metabolism is still remained unclear. In the present paper, the mechanism underlying the transcriptional regulation of SMYD3 on CYP1A1 and CYP1B1, two major phase Ⅰ metabolizing enzymes of estrogen, was investigvated. This research would provide a new theoretical basis for the understanding of the development, prevention and treatment of breast cancer and the rational use, R&D of drugs.Firstly, the human breast cancer cell line MCF-7 was treated with different concentrations of 17β-estradiol (E2), and the results of RT-PCR and western blot showed that the expression of CYP1A1 and CYP1B1 could be dose-dependently upregulated by E2. When the expression of endogenous SMYD3 was specificly supressed by RNA interference, the transcription and expression of CYP1A1 and CYP1B1 was further increased significantly. Besides, the results of high performance liquid chromatography (HPLC) analysis also showed that the down-regulation of endogenous SMYD3 might result in the decrease of residue E2 in the culture medium of cells, and the increase of 2OH-E2 and 4OH-E2, the metabolites of E2, in the cells. In addition, the analysis of Real time RT-PCR and immunohistochemistry showed that the expression of SMYD3 in cells and tissues of breast cancer was significantly higher than that in normal breast cells and cancer adjacent tissues, whereas the expression of CYP1A1 and CYP1B1 exhibited a significant negative correlation with the expression of SMYD3. Taken together, these results indicated that the expression of SMYD3 could inhibit the transcription of CYP1A1 and CYP1B1, and therefore suppress the metabolism of estrogen.Next, based on the bioinformatics analysis, we found that both the promoter of CYP1A1 and the promoter of CYP1B1 contained several SMYD3 binding elements (SBE), suggesting that they might be potential target genes for SMYD3 transcriptional regulation. The results of luciferase reporter assay and chromatin immunoprecipitation (ChIP) analysis showed that SMYD3 could associated with SBEs located at -281 site in the CYP1A1 promoter and -21 site in the promoter of the CYP1B1, and the level of histone H4K.20 methylation around these two site is also increased. Furhtermore, when SMYD3△NHSC, a HMT-activity-depleted mutant of SMYD3, was transfected into MCF-7 cells, real time RT-PCR analysis showed that the inhibitory effect of SMYD3 on CYP1A1 transcription was eliminated, while the inhibitory effect on CYP1B1 transcription was just partially weakened. These results indicated that H4K20 methylation might be essential to the suppress effect of SMYD3 on CYP1A1, whereas there might still be other mechanisms underlying the inhibitory effect of SMYD3 on the transcription CYPIBI except for the H4K20 methylation.Combined with our previous results of miRNA microarray and bioinformatics analysis, we found that there is a miR-200c binding sites (MRE) existed in the 3’UTR of CYP1B1, and miR-200c might be upregulated by SMYD3. Using luciferase reporter analysis, RT-PCR and western blot, we confirmed that miR-200c could suppress the transclation of CYP1B1 via association with the MRE in the 3’UTR of CYP1B1. In addition, with the decrease of the concentration of E2, expression of miR-200c in MCF-7 cells was significantly increased, while the CYPIBI was markedly reducd, suggesting that there is a negative correlation between miR-200c and CYPIBI in MCF-7 cells, miR-200c might play an important role in the regulation of E2 and SMYD3 on the transcription of CYP1B1. |
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