In vitro transfection of manganese superoxide dismutase small interfering rna suppresses stemness of human breast cancer stem cells (aldehyde dehydrogenase 1-positive): Focus on OCT4 mRNA expression and mammosphere-forming capacity

Introduction: Aldehyde dehydrogenase 1-positive (ALDH1+) breast cancer stem cells (BCSCs) are a small population of tumor cells with high capacity of tumorigenicity and oxidative stress. Manganese superoxide dismutase (MnSOD) is specifically expressed in mitochondria as the primary defense against superoxides, which are one of the causes of oxidative stress in cells. The aim of this study was to determine the impact of suppressing MnSOD expression using small interfering RNA (siRNA) on the stemness, tumorigenicity, and viability of BCSCs. Materials and Methods: In vitro transfection of ALDH1+ BCSCs was performed using 33 and 66 μM specific MnSOD siRNA under standard culture conditions. Total RNA and protein were extracted from the transfected cells using TriPure® Isolation Reagent and RIPA® lysis buffer. Cell viability was measured using a trypan blue exclusion assay. The relative expression of MnSOD and OCT4 mRNAs was analyzed using one-step quantitative reverse transcription polymerase chain reaction. MnSOD activity was determined by xanthine oxidase inhibition assay (RanSOD® kit). Cellular superoxides were measured using a dihydroethidium assay, and tumorigenicity was observed with mammosphere-forming unit. Results: After siRNA incubation for 48 h, MnSOD was suppressed by 0.176-fold (P < 0.01), MnSOD enzyme-specific activity was reduced 70.4%, cellular superoxide levels increased by 1.13-fold, OCT4 expression was suppressed by 1.98-fold (P < 0.05), and mammosphere-forming unit decreased by 36.5% (P < 0.05) compared with the corresponding negative controls. The viability of the ALDH1+ BCSCs was reduced 75% (P < 0.05). Conclusion: Our results suggest that suppression of MnSOD expression may be a promising target to reduce stemness and tumorigenicity of ALDH1+ BCSCs.