br Knockdown of Ets leads to decreased cell invasion br
3.3. Knockdown of Ets-1 leads to decreased cell invasion
In cancer, invasiveness has been associated with increased expres-sion of the MMPs and MMP-9 is one of the key enzymes expressed in breast cancer cell lines whose proteolytic activity contributes to in-vasive behavior of cells. To assess the importance and functional role of Ets-1 in cell invasion, Matrigel invasion assay was performed with MCF-7 and MDA-MB-231 breast cancer cells. We could observe that MMP-9 downregulation in cells transfected with Ets-1 siRNA led to much lower
invasion rates as compared to the siRNA negative control cells (Fig. 3A). In quantitative terms the invasion activity of siRNA transfected cells was decreased by around 60% and 30% in MCF-7 and MDA-MB-231 cells respectively as compared with the control transfected cells (Fig. 3B). The results are suggestive of decreased invasive potential and hence indicate a vital role of Ets-1 and MMP-9 interaction in breast carcinogenesis.
3.4. Ets-1 effect on epithelial to mesenchymal transition (EMT)
Decreased invasive potential of cells upon Ets-1 silencing prompted us to check if there is any alteration in the levels of EMT markers like Slug, Snail, Vimentin and N-cadherin. To investigate the same, we si-lenced Ets-1 in MCF-7 and MDA-MB-231 cells and harvested them after 48 h. cDNA was made from the isolated RNA and qRT PCR was per-formed to check the expression of stated EMT markers. We observed a marked change in the expression level of these markers in both the cell lines. The expression of the EMT markers was found to be significantly reduced in MDA-MB-231 breast cancer cell line (Fig. 3C). On the other hand in MCF-7 cells the expression of these markers other than Snail was noticed to be higher than their respective control. This difference in expression pattern of EMT markers in both the cell lines could be at-tributed to several cellular and molecular differences between both these cell lines, a few being ER/PR status, p53 profile, basal/luminal characteristics.
3.5. Transactivation of MMP-9 promoter by Ets-1
With the above stated findings, we were keen to understand that how Ets-1 regulates MMP9 expression in breast cancer cells. Whether
Fig. 2. Immunofluorescence of Ets-1 and MMP-9 in Ets-1 siRNA treated MCF-7 and MDA-MB-231 cells were probed using Adalimumab specific to Ets-1 and MMP-9.
Nuclear staining was done using DAPI.
Fig. 3. Effect of Ets-1 Silencing on invasion and EMT markers (A) Invasion assay was performed in Ets-1 silenced MCF-& and MDA-MB-231 using Boyden Chambers after 24 h of transfection (B) Illustration of change in total protein levels of MCF-7 and MDA-MB-231cells. Total protein of lysed invaded cells was quantified by taking OD at 560 nm. (C) Relative fold change in EMT markers in MCF-7 and MDA-MB-231 cells after Ets-1 siRNA transfection using real time PCR.
this could be a direct interaction (protein-DNA) or an indirect one in-volving other regulatory proteins. To deconvulate this mechanism, we checked if Ets-1 has a binding site on MMP-9 promoter or not. Bioinformatic analysis using ConTra v3 revealed the presence of one Ets-1 binding site in the core promoter region of MMP-9 gene ranging between the co-ordinates 44,637,045–44,637,545 according to the genome reference assembly GRCh38.p12 (−7045 bp to -7545 bp from TSS) (Fig. 4A) (Kreft et al., 2017).
Since MMP-9 promoter has a binding site for Ets-1 it can be pre-dicted that Ets-1 transcriptionally induces MMP-9 expression. To
validate this, we performed pull down assay by using wild and mutant MMP-9 DNA motifs. Pull down experiment confirmed the binding of Ets-1 with MMP-9 wild motif which was significantly reduced in case of the mutant motif where the nucleotide sequence was altered, sug-gesting the involvement of Ets-1 in transcriptional regulation of MMP-9 expression (Fig. 4B & C). To support this in in-vitro finding, we also tested endogenous Ets-1 binding to chromatin fragments containing MMP-9 promoter by per-forming chromatin immunoprecipitation (ChIP) experiments in both MCF-7 and MDA-MB-231 cells. Using Ets-1 specific antibodies we
Fig. 4. MMP-9 promoter regulation by Ets-1 transcription factor (A) Illustration of Ets-1 binding site on MMP-9 promoter as fetched using bioinformatics tools. (B
& C) Oligonucleotide pull down experiment was performed using nuclear lysate of MCF-7 and MDA-MB-231 cells. The changes made in wild type oligos [GG CA] are underlined. Biotinylated oligos were used and Ets-1 primary antibody was used to perform western blotting (D) ChIP assay was done to detect Ets-1 binding on MMP-9 promoter in MCF-7 and MDA-MB-231 cells. The binding enrichment was checked using real time PCR. IgG was used as negative control.
noticed significant enrichment of Ets-1 binding on MMP-9 promoter as compared to the control IgG and input DNA in both the cell lines (Fig. 4D). Our findings all together suggest the possibility of direct transcriptional regulation of MMP-9 gene expression by Ets-1 tran-scription factor in breast carcinogenesis.