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  • Gene br Oligonucleotide pull down assay br Pull


     Gene 711 (2019) 143952
    2.7. Oligonucleotide pull down assay
    Pull down assay was performed by extracting nuclear protein from MCF-7 and MDA-MB-231 Tigecycline using nuclear extraction kit (Thermo Scientific, USA). 3 μg of biotinylated MMP-9 wild motif, MMP-9 mutant motif and 300 μg of nuclear extract were incubated for 2 h at room temperature in Ix PBS binding buffer. The whole complex was then incubated with 40ul streptavidin resin (G-Biosciences) overnight in 100ul 1× PBS binding buffer at 4 °C. Next day the whole complex in-cluding streptavidin resin was centrifuged at 2000 rpm for 10 min at 4 °C. The supernatant was removed and the resin/protein complex was boiled with SDS PAGE loading buffer and was proceeded as in western blotting analysis using Ets-1 antibody (ab26096, Abcam).
    ChIP (Chromatin immunoprecipitation) assays were performed as per instructions of the ChIP Assay Kit provided by EpiGenetek, USA. 3 × 106 cells of MCF-7 and MDA-MB-231 were cultured in 6 well plates and were fixed with 1% formaldehyde for 10 min followed by quenching with 1.25 M glycine and then lyse. Chromatin was sheared to an average size of 500 bp using a Q Sonica (Model-Q125) sonicator with 30 cycles of 20 s on and 20 s off each. 100ul of cell lysate was used to isolate input DNA as per manufacture instructions and ChIP was per-formed using 300ul of the sonicated cell lysate by incubating it with 10 μg of Ets-1 antibody overnight at 4 °C. Mouse IgG was used as an isotype control. The eluted ChIP-DNA was purified and analyzed for MMP-9 promoter enrichment by SYBR green real time PCR using the following primers for MMP-9: forward 5′- CTGCTGTTTTCTAGAGGC TGC -3′ and reverse 5′- CCTTTTGCAACACCCCCTC -3′. The results were then expressed as fold change over control.
    3. Results
    3.1. Ets-1 gene expression was repressed efficiently and specifically after the transfection of siRNA
    Our previous findings have confirmed that Ets-1 is overexpressed in breast cancer tissue samples and have observed a differential expression pattern between receptor positive and negative status in these breast cancer patients (Nazir et al., 2019). The results prompted us to further explore the potential role and underlying mechanism of Ets-1 in breast cancer cells MCF-7 (ER, PR +ve, Her2 -ve) and MDA-MB-231 (ER, PR and Her2 -ve) and were analyzed by western blotting experiments to check the expression profile of Ets-1. We could observe a differential expression of Ets-1 in both MCF-7 and MDA-MB-231 breast cancer cell lines. However, the expression pattern of Ets-1 was higher in MDA-MB-231 breast cancer cells as compared to MCF-7 cells (Fig. 1A).
    Further, transfection of predesigned Ets-1 siRNA at three different concentrations (35, 70 and 90 pmole) in both the MCF-7 and MDA-MB-231 breast cancer cell lines, resulted in downregulation of Ets-1 ex-pression, as observed by semi-quantative PCR. Ets-1 was significantly downregulated in MDA-MB-231 cell line with 35 pmole of transfected siRNA. Increase in the dose of siRNA further downregulated the ex-pression of Ets-1 and the highest efficiency was achieved at 90 pmole post 48 h of transfection. In case of MCF-7 Ets-1 expression started to decrease at 35 pmole transfection but surprisingly increased at 70 pmole and again decreased at 90 pmole siRNA concentration. So, the maximum reduction in the expression levels of Ets-1 was found at 90 pmole siRNA concentration in transfected cells of both the cell lines (Fig. 1B). Therefore, the maximum downregulation of Ets-1 in both the MCF-7 and MDA-MB-231 cell lines was observed at 90 pmole con-centration when compared to control siRNA which was further used for all the experiments hereafter.
    Fig. 1. Ets-1 siRNA transfection and effect on MMP-9 expression (A) Basal expression of Ets-1 in MCF-7 and MDA-MB-231 breast cancer cell lines. (B) Ets-1 expression checked by semi-quantative PCR after Ets-1 siRNA transfection at 35, 70 and 90 pmole for 48 h in MCF-7 and MDA-MB-231 breast cancer cell lines. Β-actin was used as an endogenous marker. (C) Ets-1 and MMP-9 protein expression was checked using western blotting experiment after Tigecycline silencing of Ets-1 siRNA in MCF-7 and MDA-MB-231 cells for 48 h (D) Relative fold change in mRNA expression of Ets-1 and MMP-9 expression by real time PCR experiments of the same cell lines.
    3.2. Ets-1 knockdown resulted in down regulation of MMP-9 expression r> As MMP expression correlates with invasive behavior in advanced breast cancer, we examined expression profile of MMP-9 after Ets-1 siRNA treatment. Also, MMP-9 and Ets-1 have been shown to work in synchronization in ovarian cancer. This documented literature moti-vated us to check this interaction in breast carcinogenesis. The cells treated with 90 pmole Ets-1 siRNA for 48 h showed significant down regulation of MMP-9 in both MCF-7 and MDA-MB-231 breast cancer cell lines as analyzed by western blot experiments (Fig. 1C). In order to further validate whether Ets-1silencing leads to down regulation of MMP-9 mRNA expression, real time PCR was also performed. In real time experiments, the expression of Ets-1 and MMP-9 was significantly reduced in both MCF-7 and MDA-MB-231 compared to the negative control transfected ones (Fig. 1D). These results suggested that Ets-1siRNA significantly down regulate MMP-9 expression at transcrip-tional and translational level. Similarly, in consistence with the western blotting and real time results, immunofluorescence also showed a considerable decrease in MMP-9 expression after Ets-1siRNA transfec-tion (Fig. 2).