Differential sequences of exosomal NANOG DNA as a potential diagnostic cancer marker

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.

Differential sequences and single nucleotide polymorphism of exosomal SOX2 DNA in cancer

“…However, since the primer pair did not yield a PCR product with cytoplasmic or genomic DNA template either, we do not have a confirmation that this part of the SOX2 gene is missing in the exosomes. Single Nucleotide Polymorphism (SNP) in exosomal SOX2: The PCR product sequences of SH-SY5Y, GBM and CD133 + GBM derived exosomes when compared to that of NSC derived exosomes or the NCBI reported SOX2 gene sequence did not reveal obvious differences such as insertions/deletions of multiple nucleotide or DNA fragments as found in NANOG and NANOGP8 [10]. However, SNP was observed in the exosomal PCR products, cancer as well as control, in varying degrees.…”

“…We transfected HEK293 cells with SBI's XPack MSCV-XP-RFP-EF1α-Puro Expression Lentivector (catalog # XPAK731PA-1) that uses an optimized XPack exosome targeting tag to package RFP into exosomes. The RFP containing exosoms were stained with green fluorescent, lipophilic carbocyanine DiO dye and the stained exosomes were imaged using the Zeiss 710 with the Zeiss AxioObserver microscope [10]. The exosomes were further purified with magnetic beads conjugated with the antibody for exosome cell surface marker CD63 and directly used as templates without further extraction of DNA [28].…”

“…Therefore, the confocal images confirm the presence of exosomes in the sample. [10]. In the current study, we are extending our investigation to another exosome associated embryonic stemness gene, SOX2 (Sex-determining region Y (SRY)-box 2 (SOX2) [Gene ID: 6657, updated on 4-Aug-2019].…”

DNA Associated with Circulating Exosomes as a Biomarker for Glioma

“…DNase treatment of the intact exosomes reduces the total DNA yield significantly, proving the presence of the surface-associated DNA population [16]. Irrespective of their location, cancer exosomes carry single and double-stranded DNA molecules of various types that include tumor-specific oncogene amplification, aberrated chromosomal DNA, nuclear DNA, micro-nuclear genomic DNA, ribosomal DNA, cell-free DNA (cfDNA), cDNA, mtDNA as well as elevated levels of retrotransposon elements [8,15,26,27]. The type of DNA to be loaded to an exosome is affected by several factors, including the cell treatment.…”

“…The size of this insert is also significant, indicating its prospective role as a microRNA-annealing site. The microRNAs are typically 19-25 nucleotides long and their annealing to 3 UTR region of an mRNA has an expression regulatory role in cancers [27,33]. This study needs further investigation-using cancer exosomes derived from body fluids of enough number of patients-to gain statistical power.…”