br Ets transcription factor regulates the expression
Ets-1 transcription factor regulates the Ciraparantag of matrix de-grading proteinases called matrix metalloproteinase (MMPs). These are also called matrixins and includes a family of zinc-enriched en-dopeptidases which are initially expressed as inactive pro-enzymes but due to proteolytic processing and/or degradation become active en-zymes and impart their important role in extra-cellular matrix-model-ling in development, wound healing, inflammation and cancer (Stamenkovic, 2003; Parks et al., 2004). In cancer microenvironment, MMPs play a pivotal role in the initiation, development and progression of cancer via various mechanism(s) (Kessenbrock et al., 2010; Gialeli et al., 2011). One of the key member of MMPs known to play an es-sential role in cancer is matrix metalloproteinase-9 (MMP-9), also known as gelatinase B has the potential in the degradation of two basic components of basement membrane i.e. denatured collagen and col-lagen type IV. Its overexpression pays the way for tumor progression and metastasis (Mehner et al., 2014). Strong association in the ex-pression of MMP-9 with aggressive and metastatic breast carcinogenesis has been found to be among the 70 genes in the Rosetta signature for poor prognosis of breast carcinogenesis (van 't Veer et al., 2002). Dif-ferential role of MMP-9 has been found in various processes of tumor development which include invasion, induction of angiogenesis and immunomodulation of microenvironment associated with tumor. Fur-ther, its substantial role in the creation of pre-metastatic niche that promotes colonization to other organ sites has been established (Kessenbrock et al., 2010). Higher levels of MMP-9 have been asso-ciated with increased invasiveness, metastasis and poor prognosis in cervical (Yu et al., 2009), colorectal (Zeng et al., 1996), ovarian (Sillanpaa et al., 2007) and in breast cancer (McGowan and Duffy, 2008). In addition, elevation in the expression level of MMP-9 in serum and urine has shown its association in the metastatic and prognostic analysis of a variety of tumors (Roy et al., 2009). r> In view of the above, the present study was designed to investigate the role and regulation of Ets-1 and MMP-9 in the invasion and pro-gression of breast carcinogenesis as MMP-9 is associated with various pathways governed by different genes and transcriptional factors in various cancers (Zhang et al., 2014).
2. Materials and methods
The human breast cancer cell lines MCF-7 and MDA-MB-231 were procured from American Type Culture Collection (ATCC), USA and
Primer sequence used in real time PCR.
cultured in Dulbecco’s modified eagle medium low glucose (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) at 370C in 5% CO2.
2.2. siRNA transfection followed by semi and quantitative real-time PCR
MCF-7 and MDA-MB-231 breast cancer cells (3 × 105) were plated in 6-well plates and were allowed to adhere for 24 h before transfection experiments. For transfection, 3 μl of Lipofectamine™ 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) and 90 pmol of Ets-1 siRNA (Sigma-Aldrich, USA) solution were mixed with opti-MEM, with a final volume of 200 μl for each well. Before transfection different amounts of Ets-1 siRNA (35, 70 and 90 pmole) were checked to achieve the op-timum dose needed for transfection. Gentle mixing of the complex was followed by incubation at room temperature for 30 min and was added to each well containing 800 μl opti-MEM. After 4 h, transfection media was replaced with complete DMEM and was incubated for 48 h under normal cell culture conditions. Transfected negative control containing negative control siRNA (Sigma-Aldrich, USA) was prepared for both the cell lines.
2.3. RNA extraction, cDNA synthesis, semi-quantitative and quantitative real-time PCR
Total cellular RNA was isolated from cultured cells using TRIzol® Reagent (Sigma-Aldrich, USA) according to the manufacturer’s in-structions. RNA quantification was done using Qubit 3.0 Fluorometer (Thermo Fisher, USA) and 2μg of RNA was used for cDNA preparation and synthesis using iScript™ cDNA Synthesis Kit (BIO-RAD). The quality of RNA extracted was also visualized on ethidium bromide stained 2% agarose gel. Semi-quantitative PCR was carried out using Ets-1 specific primers. The amplification reaction was carried out in 25ul reaction volume in 0.2 ml PCR tubes. The reaction contains 2.5ul of 10× reac-tion buffer, 1ul of 10 mM of each primer, 1ul of 10 mM dNTPs, 2.0ul template cDNA and 0.2ul of 5 U/ul of Taq (Thermo Scientific). The PCR reaction consisted of an initial melting step of 95 °C for 5 min; 32 cycles at 95 °C for 30 s, at 58 °C for 35 s and at 72 °C for 30 s and a final elongation step of 72 °C for 10 min. PCR product was then visualized on ethidium-bromide stained 2% agarose gel.