The impact of HPV16 and HPV18 E6/E7 oncogenes on gene expression in non-tumorigenic keratinocytes (HaCaT cells)

This analysis aimed to identify differentially expressed genes in non-tumorigenic keratinocytes transduced with E6/E7 oncogenes of HPV16 or 18. The working cell models were named HaCaT E6/E7 HPV16, HaCaT E6/E7 HPV18, and HaCaT pLVX (empty lentiviral vector) as controls. mRNA 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 bioinformatics pipeline analysis was executed as described below: Rstudio (v2023.06.1+524) and Galaxy (https://www.usegalaxy.orgaccessed on 15 August 2023) open-source platforms were used to analyze the Illumina raw data. The Quality Check was performed through the FastQC tool (v0.12.1); 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.p14 v43). The resulting 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 DESeq2 tool (v2.11.40.8). 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 chemokine genes with an adjusted p-value (adj p) ≤ 0.05 and fold change (Log2FC) > 1 .5 or < -1.5 were selected as differentially expressed genes (DEGs). Some genes from the resulting outputs were validated through qPCR. Conclusions: Our study found 3296 differentially expressed genes in HaCaT E6/E7 HPV16 and 1943 in HaCaT E6/E7 HPV18. These results suggest that E6/E7 oncogenes from HPV16 and 18 can broadly modify transcriptomic expression. Total RNA extraction was achieved on transduced keratinocytes infected with lentiviral particles that bear the E6/E7 genes of HPV16 or 18; this process was addressed in duplicate. The keratinocytes transduced with the empty pLVX vector were used as calibrator in the comparison of each of the cell models. After that, the two replicates were compared as a single end of each model vs. the control for the development of the differential expression analysis.