Abstract
NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. As the roles of these genes are intimately involved with glioblastoma multiforme progression and exosomes are critical in intercellular communication, we conducted a detailed analysis of the association of the NANOG gene family with exosomes to identify diagnostic markers for cancer. Exosomes were precipitated from conditioned culture media from various cell lines, and NANOG gene fragments were directly amplified without DNA isolation using multiple primer sets. The use of the enzymes AlwNI and SmaI with restriction fragment length polymorphism analysis functioned to distinguish NANOGP8 from other NANOG family members. Collectively, results suggest that the NANOG DNA associated with exosomes is not full length and that mixed populations of the NANOG gene family exist. Furthermore, sequence analysis of exosomal DNA amplified with a NANOGP8 specific primer set frequently showed an insertion of a 22 bp sequence into the 3’ UTR. The occurrence rate of this insertion was significantly higher in exosomal DNA clones from cancer cells as compared to normal cells. We have detected mixed populations of NANOG DNA associated with exosomes and have identified preferential modulations in the sequences from cancer samples. Our findings, coupled with the properties of exosomes, may allow for the detection of traditionally inaccessible cancers (i.e. GBM) through minimally invasive techniques. Further analysis of exosomal DNA sequences of NANOG and other embryonic stemness genes (OCT3/4, SOX2, etc.) may establish a robust collection of exosome based diagnostic markers, and further elucidate the mechanisms of cancer formation, progression, and metastasis.
Highlights
Exosomes are extracellular microvesicles (30–100 nm) released by almost all types of cells upon fusion of its multi-vesicular body with the plasma membrane
Colorization of red fluorescent protein (RFP) on the inside and DiO green fluorescence on the outside of the exosomal membranes was observed by confocal microscopy, confirming the presence of intact exosomes in our samples preparation (Fig 1A, 1B and 1C)
The identities of the PCR products were confirmed through restriction fragment length polymorphisms (RFLP) analysis by the identification of AlwNI or SmaI digestion (Fig 3)
Summary
Exosomes are extracellular microvesicles (30–100 nm) released by almost all types of cells upon fusion of its multi-vesicular body with the plasma membrane. Known for their role in cell to cell communication, exosomes have demonstrated an ability to unload their contents and contribute to the transformation of normal and stem cells to cancerous states [1, 2]. 6901-7005), Antibody targeting of IL1RAP and studying their therapeutic effects in mouse models of Alzheimer’s disease, Principal Investigator, (KS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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