Silencing of NILCO by Small Interfering RNA Effects MDR1 in Colon Cancer Cells

Abstract

The aim of the study is to investigate the effect of silencing of NILCO by siRNA on the MDR1 gene, which plays a role in chemoresistance. Cells of the human colon cancer lines Caco‐2 and HT‐29 were cultured at 37˚C in DMEM supplemented with 5% FBS in a humid incubator with 5% CO2. Exponentially growing untreated Caco‐2 and HT‐29 cells were collected and plated (4 x 105/ well in 2 mL) 24 hours before transfection. Plated cells were treated with either double‐stranded siRNA targeting Notch1 mRNA, leptin mRNA, IL‐1 β mRNA, control siRNA, or Paclitaxel (PTX) alone, Notch1 siRNA+PTX, Leptin siRNA+PTX, IL‐1β siRNA+PTX. The siRNAs of Notch1, IL‐1β and Leptin, were applied to the cells at a final concentration of 30 nM, and the cells were transfected with siRNA for 48 hours. After transfection, 100 nM Paclitaxel chemotherapeutic agent for HT‐29 and 130 nM for Caco‐2 was applied to the cells. Cells were harvested for PCR analysis after treatment with PTX for 24 hours.According to our findings, MDR1 gene expression was significantly increased in the paclitaxel group compared to the control group (p<0.05). Silencing of Notch, leptin and IL‐1β decreased MDR1 gene expression compared with the control group, but it was not statistically significant. (p>0.05). However, our results indicated that Notch1 siRNA+PTX, IL‐1β siRNA+PTX and Leptin siRNA+PTX downregulated MDR gene expression in the Caco‐2 cell line compared with PTX alone treated group (p<0.01). In addition, MDR1 gene expression levels were significantly decreased in the Leptin siRNA+PTX and IL‐1β siRNA+PTX groups compared with PTX alone group in the HT‐29 cell line, but the decrease in the Notch1 siRNA+PTX group was not statistically significant. (p<0.01, p<0.05, p>0.05). In conclusion, knockdown of NILCO genes is effective in regulating MDR1 expression, which is a hallmark in chemoresistance.The present study was supported by Eskisehir Osmangazi University Scientific Research Projects (BAP) Coordination Unit (grant no. 202011D11).