Exosomes play a key role in cell-to-cell communication and are integral component of the tumor microenvironment. Recent observations suggest transfer of RNA through tumor-derived exosomes that can potentially translate into regulatory proteins in the recipient cells. Role of cervical cancer-derived exosomes and their transcript cargo is poorly understood.
Researchers from the University of Delhi extracted the total RNA of exosomes from HPV-positive (SiHa and HeLa) and HPV-negative (C33a) cervical cancer cell lines and estimated the transcripts using Illumina HiSeq X. Further, validation of HPV transcripts were performed using RT-PCR. 3099 transcripts were found to be differentially-exported in HPV-positive vs. HPV-negative exosomes (p value <0.05). Analysis of top 10 GO terms and KEGG pathways showed enrichment of transcripts belonging to axon guidance and tumor innervation in HPV-positive exosomes. Among top 20 overexpressed transcripts, EVC2, LUZP1 and ANKS1B were the most notable due to their involvement in Hh signaling, cellular migration and invasion, respectively. Further, low levels of HPV-specific reads were detected. RT-PCR validation revealed presence of E6*I splice variant of HPV18 in exosomal RNA of HeLa cells. The E6*I transcripts were consistently retained in exosomes obtained from HeLa cells undergoing 5-FU and cisplatin-induced oxidative stress.
Differentially expressed transcript profile of cervical cancer exosomes
A Distinct mRNA expression profiles in HPV-positive vs. HPV-negative and HPV16-positive vs HPV18-positive. The inclusion criteria of these transcripts was a 2-fold difference of log2 (fold-change) in either direction with a p value <0.05. Red signal, represents higher relative expression as compared to green signal. B Quantitative Venn diagram showing differentially expressed gene. C Volcano plot showing overall clustering of differentially expressed exosomal transcripts in HPV-positive vs. HPV-negative and HPV16-positive vs. HPV18-positive (left panels). Volcano plot showing distinct clustering of top 20 transcripts (right panels). Blue color indicates log2 fold change ≥1.5 and p value ≤0.05. Red dots indicate absolute log2 fold change ≥1.5 and FDR/adjusted p value ≤0.05. Gray lines indicate the marginal lines separating DEGs from non-DEGs, with the horizontal lines denoting the p value threshold (p ≤ 0.05) and vertical lines the fold change cut off (≥ 1.5 or < 0.67).
These data suggest the enrichment of poly-A RNA transcripts in the exosomal cargo of cervical cancer cells, which includes pro-tumorigenic cellular RNA and viral transcripts such as HPV E6, which may have clinical utility as potential exosomal biomarkers of cervical cancer.