Transforming properties of E6/E7 genes from Beta HPV80 in a fibroblast model in vitro

This analysis aimed to elucidate the effect of transduced E6/E7 HPV80 genes in primary fibroblasts employing lentiviral vectors, examining their influence on the transcriptome of our cellular model and demonstrating their capacity to prolong the lifespan of the culture and lead to malignant transformation. Total RNA sequencing was performed on the Illumina NovaSeq 6000 platform by Novogene Bioinformatics Technology Co., Ltd, Beijing, China, with two independent replicate sequences for each cell model. Methodology: The execution of the bioinformatics pipeline analysis was carried out as follow: R (ver. 4.4.1), RStudio (2024.09.0+375) and Galaxy (https://www.usegalaxy.org accessed on 04 September 2024) open-source platforms were used to analyze the Illumina raw data. The Quality Control was conducted through the FastQC tool (v0.74+galaxy0). Afterward, all the reads were processed by the Rsubread package (v2.14.2); at that point, the reads were trimmed and aligned to the Human Genome Reference (GRCh38.p13 v46). The obtained output was the BAM files, of which the number of reads was counted by the FeatureCounts tool (v2.0.3); at the final step, the Differential Expression Analysis was settled by the DESeq2 package for R (ver. 1.42. 0). The differential gene expression measurements were normalized by DESeq2's median of ratios (median of ratio of gene counts relative to geometric mean per gene) method. All genes with an adjusted p-value (p-adj) minus or equal to 0.05 and fold change (Log2FC) up to 2 or less than minus 2 were selected as differentially expressed genes (DEGs). Conclusions: Our study found that E6/E7 HPV80 genes have the capacity to prolong the lifespan of the primary Fibroblasts mainly throug the disregulation of pathways related to cell cycle progression, p53-related DNA repair machinery and cell cycle arrest, apoptosis, immune evasion, cell growth, differentiation, and cell death. Primary Fibroblasts that constitutively expressed E6/E7 HPV80 genes (transduced with lentiviral vectors harboring E6/E7 HPV80 genes) underwent total RNA extraction (process was addressed in duplicate). After that, the replicates were compared vs. the controls (pLVX vector alone) for the development of the differential expression analysis.