br A significant co relation of Ets with breast

A significant co-relation of Ets-1 with breast cancer tumor samples was observed in our recent finding (Nazir et al., 2019). The present study was performed to investigate the regulatory role of Ets-1 tran-scription factor in breast cancer along with its downstream target gene MMP-9 by utilizing RNA interference in breast cancer cell lines MCF-7 and MDA-MB-231. We observed a differential expression of Ets-1 in these cell lines. MCF-7 cell line being less invasive and less aggressive showed lower expression of Ets-1 in comparison to MDA-MB-231 which
belongs to the triple negative breast cancer cell line and is more in-vasive and more aggressive. To check the role of Ets-1 in these cell lines we knocked down the Ets-1 gene by using different concentrations of Ets-1 siRNA in both the MCF-7 and MDA-MB-231 breast cancer cell lines. In case of MDA-MB-231 silencing started at 35 pmole of siRNA and the highest efficiency was achieved at 90 pmole post 48 h of transfection. However, in case of MCF-7 decreased expression of Ets-1 started at 35 pmole of siRNA showed highest effeiciency at 90 pmole but unexpectedly increased at 70 pmole. Though the reason for this phenomenon is not fully understood by us as well, however, there could be several probable reasons such as the Geneticin have a tendency to revert back to the changes introduced to them in specific ways. The MCF-7 cells might be displaying an increase in Ets-1 expression at 70pmole as some off site targets might result in the induction of some different signaling pathways and in turn activating the alternate signaling pathways. Another plausible explanation could be that Ets-1 siRNA may not work efficiently at some higher concentraation in MCF-7 cell line because of overdose/time. Also, as it is siRNA based approach it is likely possible that after transfection the cells may change their behavior or become senescent or resistant resulting in no loss of gene expression. Some biological or experimental variation could be other reason re-sponsible for this type of phenomenon.
On silencing of Ets-1, a corresponding decrease in MMP-9 expres-sion was noted both at the protein and mRNA level as analyzed by using various molecular biology techniques such as western blotting, real time PCR and immunofluorescence. These results suggested direct ef-fect of Ets-1 knockdown on MMP-9 expression. Similar results have also been observed in various other cancers such as pancreas, prostrate, ovary and colon cancer cells (Ito et al., 2004; Ghosh et al., 2012; Kato et al., 2012; Gao et al., 2014). In addition, some other studies have also shown the involvement of various other factors to be associated with MMPs production tunneled by Ets-1 (Ito et al., 2004; Park et al., 2008; Ghosh et al., 2012; Gao et al., 2014). So these studies established the fact that Ets-1 has both direct and indirect role on the expression of MMPs.
It was interesting to note the change in invasive capacity of MCF-7 and MDA-MB-231 cells upon Ets-1 perturbation. We also found a change in EMT marker expression simultaneously. Previous studies have shown that Ets-1 is not only involved in the development of var-ious tumors but is also associated with tumor invasion (Singh et al., 2002). In mouse mammary tumor (MMT) epithelial cells and in some breast cancer cell lines overexpression of Ets-1 can up regulate MMPs and result in increased cellular invasiveness (Furlan et al., 2008; Park et al., 2008). In concordance with the above documented literature, we observed a significant decrease in the invasion capacity of MCF-7 and MDA-MB-231 breast cancer cells induced by siRNA against the Ets-1 gene. Various studies have established that MMP-9 promotes key events in tumor progression and invasion. Here we found that downregulating Ets-1 results in down regulation of MMP-9, suggesting that Ets-1 pro-motes cellular invasiveness via upregulating MMP-9.