Analysis of regulatory sequences in exosomal DNA of NANOGP8

Abstract

Exosomes participate in intercellular communication by transporting functionally active molecules. Such cargo from the original cells comprising proteins, micro-RNA, mRNA, single-stranded (ssDNA) and double-stranded DNA (dsDNA) molecules pleiotropically transforms the target cells. Although cancer cells secrete exosomes carrying a significant level of DNA capable of modulating oncogene expression in a recipient cell, the regulatory mechanism is unknown. We have previously reported that cancer cells produce exosomes containing NANOGP8 DNA. NANOGP8 is an oncogenic paralog of embryonic stem cell transcription factor NANOG and does not express in cells since it is a pseudogene. However, in this study, we evaluated NANOGP8 expression in glioblastoma multiforme (GBM) tissue from a surgically removed brain tumor of a patient. Significantly higher NANOGP8 transcription was observed in GBM cancer stem cells (CSCs) than in GBM cancer cells or neural stem cells (NSCs), despite identical sequences of NANOGP8-upstream genomic region in all the cell lines. This finding suggests that upstream genomic sequences of NANOGP8 may have environment-dependent promoter activity. We also found that the regulatory sequences upstream of exosomal NANOGP8 GBM DNA contain multiple core promoter elements, transcription factor binding sites, and segments of human viruses known for their oncogenic role. The exosomal sequence of NANOGP8-upstream GBM DNA is different from corresponding genomic sequences in CSCs, cancer cells, and NSCs as well as from the sequences reported by NCBI. These sequence dissimilarities suggest that exosomal NANOGP8 GBM DNA may not be a part of the genomic DNA. Exosomes possibly acquire this DNA from other sources where it is synthesized by an unknown mechanism. The significance of exosome-bestowed regulatory elements in the transcription of promoter-less retrogene such as NANOGP8 remains to be determined.

Fig 1. Schematic of the steps in exosomal and genomic DNA sequence analysis.

Fig 2. RFLP analysis of NANOGP8 transcript.

cDNAs from NSC and GBM were amplified with NANOGP8 primers and digested with RE AlwNI. (A) Initial amplification yielding two bands of PCR products, pointed by red arrows are labeled as “U” and “L” for upper and lower bands respectively (S1 Raw image). (B) Reamplification of the upper and lower band, separately (S2 Raw image). (C) RE-AlwNI digestion of upper bands showing partial digestion, confirming the presence of a mixed population of NANOG and NANOGP8 transcripts (S3 Raw image). (D) RE-AlwNI digestion of lower bands showing complete digestion of the PCR product validates that the PCR product belongs to the NANOGP8 transcript alone. Panel D is made by removing the irrelevant lanes from S4 Raw image.

Fig 3. Quantitative PCR amplification of NANOGP8 transcript.

CD133⁺ GBM cancer stem cells have significantly higher expression as compared to the GBM primary cells comprising a mix of undifferentiated CD133⁺ CSCs and differentiated CD133^- cancer cells. CD133^- cells express negligible NANOGP8 transcript.

Fig 4. RFLP analysis of NANOGP8 gDNA PCR product.

gDNA amplified with NANOGP8 primers. PCR product digested with HpyF3I. NSC and GBM show complete digestion, affirming the presence of the NANOGP8 transcript. A PCR product of 529 bp is digested to yield fragments of 287, 234, and 8 bp each. The 8 bp fragment is not visible in the image. S5 Raw image shows the details.

Fig 5. Profiling of the core promoter motifs identified in the upstream region of NANOGP8 DNA associated with NSC-derived exosomes.

The sequences were analyzed using the YAPP Eukaryotic Core Promoter Predictor. Synergistic core promoter motifs are denoted in light gray, whereas the bars in dark grey depict individual motifs. S1A and S1B Table details the sequences and the type of promoter element.

Fig 6. Profiling of the core promoter motifs identified in the upstream region of NANOGP8 DNA associated with GBM-derived exosomes.

The sequences were analyzed using the YAPP Eukaryotic Core Promoter Predictor. The bars in dark grey depict individual motifs. No synergistic core promoter combination was detected. S2 Table details the sequences and the types of promoter elements.

Fig 7. Profiling of the core promoter motifs identified in the upstream region of NANOGP8 DNA associated with CD133^- derived exosomes.

The sequences were analyzed using the YAPP Eukaryotic Core Promoter Predictor. A singular synergistic core promoter motif is denoted in light gray, whereas the bars in dark grey depict individual motifs. S3A and S3B Table details the sequences and the type of promoter element.

Fig 8. CD133^- GBM derived exosomal DNA sequence (partial).

(A) 20 nucleotide identity to HIV-1 isolate AE-Env_CR12_Apr10R from Thailand envelope glycoprotein (env) gene (JN388157.1). (B) The nucleotides underlined in panel A show motif for a core promoter element DPE, in the HIV-1 sequence, detected by the YAPP tool. Retrovirus inserts in the upstream region may provide promoter sequences.

Fig 9. Standard BLAST analysis of NSC exosomal DNA clone after omitting the sequences for pCR4TOPO-TA vector and NANOG/NANOGP8 gDNA.

The remaining sequence reveals the identities with various retroviral integration sites.

Fig 10. Standard BLAST analysis of GBM exosomal DNA clone after omitting the sequences identical to pCR4TOPO-TA vector and NANOG/NANOGP8 gDNA.

The remaining sequence reveals the identities with HIV-1 genes.

Fig 11. Analysis of the exosomal viral identities using Multiple Sequence Alignment Viewer 1.21.0.

(A) NSC, (B) GBM—proliferating and (C) differentiating (CD133^-) GBM exosomal DNA.

Fig 12. Core promoter motifs of the NANOGP8 promoter region in CD133⁺ GBM gDNA clones analyzed using the YAPP Eukaryotic Core Promoter Predictor.

Fig 13. Analysis of the NSC and CD133⁺GBM-gDNA clone.

Detection of viral identities using Multiple Sequence Alignment Viewer 1.21.0.