Johari Behrooz, Ebrahimi-Rad Mina, Maghsood Faezeh, Lotfinia Majid, Saltanatpouri Zohreh, Teimoori-Toolabi Ladan, Sharifzadeh Zahra, Karimipoor Morteza, Kadivar Mehdi
Biochemistry Department, Pasteur Institute of Iran, Tehran. Iran.
Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran. Iran.
Anticancer Agents Med Chem. 2017;17(13):1786-1795. doi: 10.2174/1871521409666170412142507.
Myc (c-Myc) alone activates the embryonic stem cell-like transcriptional module in both normal and transformed cells. Its dysregulation might lead to increased cancer stem cells (CSCs) population in some tumor cells.
In order to investigate the potential of Myc decoy oligodeoxynucleotides for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of CSCs. To our best of knowledge this is the first report outlining the application of Myc decoy in transcription factor decoy "TFD" strategy for inducing differentiation in mESCs.
A 20-mer double-stranded Myc transcription factor decoy and scrambled oligodeoxynucleotides (ODNs) were designed, analyzed by electrophoretic mobility shift (EMSA) assay and transfected into the mESCs under 2 inhibitors (2i) condition. Further investigations were carried out using fluorescence and confocal microscopy, cell proliferation and apoptosis analysis, alkaline phosphatase and embryoid body formation assay, real-time PCR and western blotting.
EMSA data showed that Myc decoy ODNs bound specifically to c-Myc protein. They were found to be localized in both cytoplasm and nucleus of mESCs. Our results revealed the potential capability of Myc decoy ODNs to decrease cell viability by (16.1±2%), to increase the number of cells arrested in G0/G1 phases and apoptosis by (14.2±3.1%) and (12.1±3.2%), respectively regarding the controls. Myc decoy could also modulate differentiation in mESCs despite the presence of 2i/LIF in our medium the presence of 2i/LIF in our medium.
The optimized Myc decoy ODNs approach might be considered as a promising alternative strategy for differentiation therapy investigations.
Myc（c-Myc）单独激活正常细胞和转化细胞中的胚胎干细胞样转录模块。其失调可能导致某些肿瘤细胞中癌干细胞（CSCs）数量增加。
为了研究Myc诱饵寡脱氧核苷酸用于分化治疗的潜力，本研究使用小鼠胚胎干细胞（mESCs）作为CSCs模型。据我们所知，这是第一份概述Myc诱饵在转录因子诱饵“TFD”策略中诱导mESCs分化的应用的报告。
设计了一种20聚体双链Myc转录因子诱饵和乱序寡脱氧核苷酸（ODNs），通过电泳迁移率变动分析（EMSA）进行分析，并在2种抑制剂（{2i}）条件下转染到mESCs中。使用荧光和共聚焦显微镜、细胞增殖和凋亡分析、碱性磷酸酶和胚状体形成分析、实时PCR和蛋白质印迹进行进一步研究。
EMSA数据表明Myc诱饵ODNs与c-Myc蛋白特异性结合。发现它们定位于mESCs的细胞质和细胞核中。我们的结果显示，与对照相比，Myc诱饵ODNs具有降低细胞活力（16.1±2%）、增加停滞在G0/G1期的细胞数量（14.2±3.1%）和凋亡细胞数量（12.1±3.2%）的潜在能力。尽管培养基中存在2i/LIF，Myc诱饵也可以调节mESCs的分化。
优化后的Myc诱饵ODNs方法可能被认为是分化治疗研究的一种有前景的替代策略。
