E6/E7 from Beta-2-HPVs 122, 38b and 107 possess transforming properties in a fibroblast model in vitro

This analysis was performed with the aim of finding the genes differentially expressed in fibroblasts transduced with the E6/E7 genes of HPV 16, 18, 38b, 107 and 122. The name given for the samples are as follows: FB-E6E7-16, FB-E6E7-18, FB-E6E7-38b, FB-E6E7-107, FB-E6E7-122, and for fibroblasts transduced with the empty lentiviral vector pLVX, they were referred as FB-pLVX. Methods: Library construction and Illumina NovaSeq 6000 sequencing was performed by Novogene Bioinformatics Technology Co., Ltd, Beijing, China, on HPV 16, 18, 38b, 107 and 122 E6E7 Transduced Fibroblasts; three independently mRNA sequences were performed for each of the cell models. Bioinformatics analyses were performed as follows: raw reads were analyzed using the open source Galaxy platform (https://www.usegalaxy.org) and RStudio software (2021.09.0). The quality of the reads was checked using the FastQC tool, and the Trimmomatic tool was used to remove ambiguous nucleotides. Subsequently, all clean reads were mapped to the human genome reference (hg38 vs 38) using Rsubread for RStudio. Once all BAM files were obtained, reads were counted using the featureCounts tool, and gene expression analysis was performed using the DESeq2 tool. The gene expression level was normalized using the fragments per kilobase of transcript per million mapped reads (FPKM) method. Data validation was performed by real-time PCR. Conclusions: Our study represents the first analysis of genes differentially expressed by the E6/E7 proteins of HPV genus Beta-2 (38b, 107 and 122), through RNA-seq technology. This study shows the transforming potential that genotypes of the Beta-2 genus, especially HPV122, also possess. These Beta-2 HPVs can modulate some of the genes that are well known to be regulated by Alpha-HPVs 16 and 18. Total RNA extraction was performed from cell models obtained from fibroblasts infected with viral particles containing the E6/E7 genes of HPV 16, 18, 38b, 107 and 122 in triplicate. Fibroblasts transduced with HPV 16 and 18 were used as positive controls, due to their known cellular transformation properties. Primary fibroblasts transduced with the empty- pLVX-vector were used as basal controls, with which all our cell models were compared. To obtain the differential expression of the genes, a comparison of the six replicates of FB-pLVX with the three replicates of each of the cell models was performed.