Transcriptome analysis of cervical cancer exosomes and detection of HPVE6*I transcripts in exosomal RNA

Abstract

Background 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. Materials and methods The total RNA of exosomes from HPV-positive (SiHa and HeLa) and HPV-negative (C33a) cervical cancer cell lines were extracted and the transcripts were estimated using Illumina HiSeq X. Further, validation of HPV transcripts were performed using RT-PCR. Results 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. Conclusion Our data suggests 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.

Quantitative Proteomics for the Identification of Differentially Expressed Proteins in the Extracellular Vesicles of Cervical Cancer Cells

“…The EVs released by tumor cells may participate in the hallmarks of cancer via a horizontal transfer of functional biomolecules to cells in the TME [ 21 , 22 ]. An evaluation of the composition of the EVs produced by cervical cancer cells has mainly led to describing their RNA content [ 23 , 24 , 25 , 26 ], but the total protein content of these EVs remains unknown, as well as the proteins contained in these EVs with a possible function in this cancer. In this study, we analyzed the protein content of the EVs derived from cervical cancer using the HeLa cell line as a first step for detecting overexpressed molecules in the EVs from cervical cancer.…”

Untold story of human cervical cancers: HPV-negative cervical cancer

“…Exosomes carry cell-specific cargos of proteins, nucleic acids, and lipids ( 64 ). Contents of exosomes vary by cell origin and by pathological and physiological conditions of exosome-secreting cells ( 65 ). Tumor cells can secrete tumor-specific exosomes such as dysfunctional miRNAs involved in cancer pathogenesis ( 66 ).…”

Advances in Exosomes as Diagnostic and Therapeutic Biomarkers for Gynaecological Malignancies

“…For instance, Wnt2B protein from CC cells is delivered to fibroblasts in exosome form, where it induces fibroblast activation into CAFs and advances CC [62]. HPV E6 transcripts were also detected in exosomes of CC cells, which might serve as potential exosome biomarkers for CC [63]. From these instances, it could be recognized that exosomes play a huge role in supporting CC progression.…”