Simultaneous targeted inhibition of Sox2‐Oct4 transcription factors using decoy oligodeoxynucleotides to repress stemness properties in mouse embryonic stem cells

Abstract

Transcriptional master regulators like Sox2 and Oct4, which are expressed in various human tumors, have been shown to cause tumor growth promotion as well as epithelial dysplasia by means of interfering with progenitor cell differentiation. In order to investigate the potential of Sox2-Oct4 transcription factor decoy (TFD) strategy for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of cancer stem cells (CSCs). Sox2-Oct4 complex decoy ODNs (cd-ODNs) were designed according to their elements in the promoter region of Sox2 gene. DNA-protein interactions between decoy ODNs and their corresponding proteins were examined by electrophoretic mobility shift assay (EMSA). Then, decoy and scrambled ODNs were transfected into mESCs with lipofectamine under 2 inhibitors (2i) conditions. Fluorescence and confocal microscopy, cell viability, cell cycle and apoptosis analysis, alkaline phosphatase, embryoid body formation assay, and real-time PCR were used to conduct further investigations. EMSA data showed that Sox2-Oct4 decoy ODNs bound specifically to their recombinant proteins. The results revealed that the synthesized complex decoy can concomitantly target Sox2 and Oct4, which subsequently represses the stemness properties of mESCs compared to controls through decreasing cell viability, arresting cell cycle in G0 /G1 phases, inducing apoptosis, and modulating differentiation in mESCs despite the presence of 2i/LIF in cell culture. While cd-ODN strategy seems to offer great promise for cancer therapy, further studies are still required to put this powerful investigative tool in practice for a wide range of human cancers.