Cell culture
The MCF-7 and MDA-MB-231 breast cancer cells (American Type Culture Collection, Washington D.C., USA) were cultured in 25 and 75 cm2 flasks (Thermo Fisher Scientific, USA) containing Dulbecco’s modified Eagle medium (DMEM; Gibco, Paisley, Scotland, UK) without phenol red, supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin/streptomycin (Lonza, Walkersville, MD, USA), and 2 mM l-glutamine (Lonza, Walkersville, MD, USA) in an incubator at 37˚C with 5% CO2.
Gene silencing with siRNA,Total RNA isolation and cDNA synthesis
The siRNA transfection protocol of the siPORT Transfection kit (gene silencing specific to S1P1, S1P3, Kv1.3, and Kv10.1) (Ambion, Carlsbad, CA, USA) was applied to the MCF-7 and MDA-MB-231 cells. Total RNA isolation from the cells was obtained according to the kit (Macherey-Nagel, Duren, Germany). cDNA synthesis was performed from the RNAs obtained from cells. In accordance with the kit method (New England Biolabs, Beverly, MA, USA), 200 ng of the total RNA per sample, random primer, and nuclease-free water were mixed in a microfuge tube to 8 µl. Then, 10 ul of the reaction mix and 2 ul of the enzyme mix were added, and the total volume was completed to 20 µl. This was then incubated for 1 hour at 42 ° C. The enzyme was incubated for 5 minutes at 80°C for inactivation. The obtained cDNAs were stored at -20°C. In addition, lateral motility and invasion experiments were performed to investigate the effect of siRNA transfection on metastatic cell behavior.
qRT-PCR
The synthesized cDNAs were mixed using appropriate primer-probes and subjected to a real time PCR analysis to determine the Kv1.3 (Hs00704943_s1, Applied Biosystems) , Kv10.1 (Hs00924320_m1, Applied Biosystems) , S1P1 (Hs 01922614-s1, Ambion) and S1P3 (Hs 00245464-s1, Ambion) gene expression levels in the cells. The control group was used as the calibrator in the expression assay in the siRNA-transfected groups. Transfection efficacy was evaluated according to this group. 18SRNAs housekeeping gene (Hs99999901_s1, Applied Biosystems) was used as normalizers in all qPCR procedures. The Ct values were calculated using the qRT-PCR Instrument (Stratagene MX3000P Real time PCR, USA) and compared [13]. Gene expression comparisons and evaluations were analyzed on the qRT-PCR device and the relationships of the data were presented with graphs. At least three wells were loaded for each sample in qRT-PCR, and gene expressions were determined to observe the silencing by reading and associating these wells according to the dedicated software of the device.
Determination of invasion
After the siRNAs were transfected, 2x105 cells were seeded in serum matrix-coated inserts (pore width 8μm) (BD Biosciences, San Jose, CA) to determine matrix invasion. After the flasks were incubated at 37 °C for 72 hours, the cells that migrated to the bottom were stained with crystal violet and counted.
Wound healing
For the determination of the lateral wound healing (wound healing), the first width measurements of three initial scars generated by scraping the cells using a 1 ml pipette tip were recorded by a micrometer-scaled ruler for each petri dish after adhesion in the incubator for 1 h. At 24 h and 48 h, the measurement was repeated. The migration of the cells to this area was determined by measuring 45 different areas (generated by drawing a 15×3 grid on the lid of the petri dish) at each time point. Gap closure measurements of the wound width were recorded with a micrometer scale ruler on the microscope. The procedure was repeated for 24, 48 and 72 hours in the following days. The results were calculated as gap closure measurements of the wound width %.