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زهرا حسن زاده، فاطمه حسن زاده، ریتا عرب سلغار

Down regulation of HDAC1, DNMT1 and MeCP2 proteins involved in regulation of estrogen receptor α gene expression in MDA-MB 468 breast cancer cell line by siRNA Zahra Hassanzadeh1, Fatemeh Hassanzadeh1, Rita Arabsolghar1,2 * 1 Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran 2 Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran *Corresponding author: Rita Arabsolghar Email: arabsolghar@sums.ac.ir Background: Breast cancer is one of the most common oncological conditions in females. Both inherited and/or sporadic mutations within the germline or somatic cells can cause the development of breast cancer. Epigenetic changes, which can affect the expression profiles of genes, can also be implicated in the pathogenesis of breast cancer. Epigenetic modification is achieved by DNA methylation and by changes within the histone proteins. Both estrogen and ER-α itself play critical roles in the development and progression of invasive breast cancer. Expression of ER-α in tumors serves as a prognostic indicator of the effectiveness of various hormonal treatments against breast cancer. Nevertheless, a subset of breast tumors may exhibit a loss of ER expression during the course of tumor progression or may lack ER-α at the point of diagnosis. Numerous investigations have demonstrated that epigenetic alterations in mammalian cells, including the hypermethylation of CpG islands located within the ESR1 promoter, are integral to the silencing of its associated gene. We aimed, in the present study, to elucidate the roles of DNMT1, HDAC1, and MeCP2 in ESR1 silencing and, as well, to explore the possibility of the selective knockdown of these proteins in re-establishing ER-α expression in the ER-negative MDA-MB-468 human breast cancer cell line. Methods: To further explain the molecular mechanisms of the silencing of the ESR1 gene in the estrogen receptor-negative MDA-MB-468 cells, we used the downregulation approach using small interfering RNAs against DNMT1, HDAC1, and MeCP2. The results of this knockdown were analyzed based on the protein and mRNA levels using the techniques of Western blotting and RT-PCR. Furthermore, we analyzed the associations of DNMT1, HDAC1, MeCP2, and p53 with the ESR1 promoter CpG island by chromatin immunoprecipitation (ChIP). Delivery of siRNAs is a powerful tool not only in the validation of protein expression within diverse cellular backgrounds but also for the dissection of many signaling pathways and even for possible therapeutic applications. Results: ChIP experiments demonstrated that the repression complex of DNMT1, HDAC1, and MeCP2 was bound together with mutant p53 to repress ESR1 expression in estrogen receptor-negative MDA-MB-468 cells. In contrast, wild-type p53 showed binding to active ESR1 promoters in MCF-7 cells. Furthermore, ectopic expression of wild-type p53 could not reactivate the ESR1 gene in MDA-MB-468 cells. We demonstrated that the knockdown of either the DNMT1 or HDAC1 gene resulted in the dissociation of the repression complex, including HDAC1 or DNMT1, MeCP2, and mutant p53 from the ESR1 promoter in these cellular contexts. The partial demethylation of the ESR1 promoter and the reappearance of ER observed in cells with downregulated DNMT1 or HDAC1 further confirmed these results. However, our study showed that the downregulation of MeCP2 in these cells did not result in the release of DNMT1 from the ESR1 promoter and further did not allow reactivation and demethylation of the ESR1 gene. These findings confirm our hypothesis of DNMT1 and HDAC1 being critical in the repression of the ESR1 promoter and further provide evidence to suggest that mutant p53 may be a strong determinant of DNA methylation. Further, a cooperative interaction by HDAC1 within this repression complex with both DNMT1 and MeCP2 is also proposed to facilitate gene silencing. Conclusion: Silencing the genes related to DNMT1, HDAC and Mecp2 proteins by siRNA and following the ChIp test showed that the inhibitory complex including DNMT1, Mecp2 and HDAC proteins by binding to the mutant P53 in MDA_MB 468 cells has the ability to bind to the silenced ESR1 promoter. and with the silence of DNMT1 or HDAC genes or both. This inhibitory complex is separated from the ESR gene promoter, but Mecp2 gene silencing is not able to break down this complex. Also, the electrophoretic expression of wild type p53 could not follow the expression of ESR1 gene in MDA_MB 468 cells. In MCF_7 cells, which are among ER positive cells. The wild type p53 is connected to the ESR1 gene promoter, but the inhibitory complex mentioned is not connected and therefore the ESR1 gene is not turned off.

siRNA, Breast Cancer, ESR1

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حسن زاده، زهرا، حسن زاده، فاطمه، عرب سلغار، ریتا، 1404، کاهش تنظیم پروتئین های HDAC1، DNMT1 و MeCP2 در تنظیم بیان ژن گیرنده استروژن α در رده سلولی سرطان پستان MDA-MB 468 توسط siRNA، کنگره بین المللی زیست پزشکی سرطان و سلولهای بنیادی، http://conf.sums.ac.ir/fa/archive_index.php?c=HN-icsbc1&aid=1514188