mRNA sequencing of novel cell lines from human papillomavirus type-16 related vulval intraepithelial neoplasia: Consequences of expression of HPV16 E4 and E5

Abstract

Vulval intraepithelial neoplasia is a precursor of vulval cancer and is commonly caused by infection with Human Papillomavirus (HPV). Development of topical treatments for vulval intraepithelial neoplasia requires appropriate in vitro models. This study evaluated the feasibility of primary culture of vulval intraepithelial neoplasia biopsy tissue to produce cell lines for use as in vitro models. A potentially immortal cell line was produced which gave rise to three monoclonal lines. These lines were characterized for HPV genomic integration and for viral gene expression using ligation-mediated PCR and quantitative PCR. Distinct patterns of viral integration and gene expression were observed among the three lines. Integration and expression data were validated using deep sequencing of mRNA. Gene ontology analyses of these data also demonstrated that expression of the HPV16 E4 and E5 proteins resulted in substantial changes in the composition of the cell membrane and extracellular space, associated with alterations in cell adhesion and differentiation. These data illustrate the diverse patterns of HPV gene expression potentially present within a single lesion. The derived cell lines provide useful models to investigate the biology of vulval intraepithelial neoplasia and the interactions between different HPV gene products and potential therapeutic agents.

Increased methylation of Human Papillomavirus type 16 DNA correlates with viral integration in Vulval Intraepithelial Neoplasia

“…Distinct integration events were detected in clones CU-VA-9D, CU-VA-9H, CU-VI-8M and CU-VI-8Y (Table 2). Integration events were not detected in CU-VA-9A or CU-VI-8P, however CU-VI-8P carried a deletion of the E2 ORF and given the well-characterised roles of E2 protein in viral replication, genome maintenance, partition and tethering [32], it appears likely that CU-VI-8P contained integrated HPV (the presence of integrated HPV in this line was also consistent with mRNA sequencing data [26]). In most lines, DIPS and APOT identified integration in the same chromosomal location, but in CU-VA-9H the two techniques detected integration in distinct locations.…”

“…CU-VA-9 was derived from a 31-year-old woman with VAIN3. Three distinct clonal lines were derived from each biopsy via single cell cloning (described in Bryant et al [26]). Nucleic acids were extracted using the AllPrep DNA/RNA kit (Qiagen) according the manufacturer's instructions.…”

Genomic characterization of viral integration sites in HPV‐related cancers

“…Our analysis included integration sites from 33 articles, including a previous systematic review, as well as integration sites obtained from TCGA or our own unpublished data (Table ). In total, we collected information on 1,551 integration events across several cancer organ sites and cell lines (Supporting Information Table 1).…”

“…Our analysis included integration sites from 33 articles, [10][11][12][13][14][15][16][17][18][19][20][21][22][23][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] including a previous systematic review, 11 as well as integration sites obtained from TCGA or our own unpublished data (Table 1). In total, we collected information on 1,551 integration events across several cancer organ sites Table 2).…”

Surface modification of tri-calcium phosphate nanoparticles by DOPE and/or anti-E6 antibody to enhance uptake of antisense of E6 mRNA

“…Carboxylated polyethylene glycol (C-PEG), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), anti-E6 antibody, DOPE, fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), isopropanol, Hank's balanced salt solution (HBSS), and RPMI 1640 medium were obtained from Sigma-Aldrich company, Germany. Single-stranded oligonucleotide (5 -CTAGCTGCAAAC-AACTATACAT-3 ) [21] was purchased from Takapo Zyst Company, Iran. The oligonucleotide was labeled with Hexachloro-fluorescein (HEX) tag.…”