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. Overall design: 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.

本分析旨在筛选经人乳头瘤病毒（HPV）16、18、38b、107及122型E6/E7基因转导的成纤维细胞中的差异表达基因。样本命名规则如下：转导上述E6/E7基因的成纤维细胞分别命名为FB-E6E7-16、FB-E6E7-18、FB-E6E7-38b、FB-E6E7-107、FB-E6E7-122；转导空慢病毒载体pLVX的成纤维细胞命名为FB-pLVX。

方法：文库构建及Illumina NovaSeq 6000测序由中国北京诺禾致源生物信息科技有限公司完成，针对HPV 16、18、38b、107、122型E6/E7转导的成纤维细胞开展；每种细胞模型均设置3次独立的mRNA测序重复。生物信息学分析流程如下：使用开源Galaxy平台（https://www.usegalaxy.org）及RStudio软件（2021.09.0）处理原始reads；通过FastQC工具评估reads质量，并利用Trimmomatic工具去除模糊碱基。随后，使用RStudio中的Rsubread工具将所有clean reads比对至人类参考基因组（hg38 vs 38）。获得所有BAM文件后，使用featureCounts工具进行reads计数，并通过DESeq2工具开展基因表达分析。基因表达水平采用每百万映射reads中每千碱基转录本的片段数（FPKM）方法进行标准化。通过实时定量PCR完成数据验证。

结论：本研究首次通过RNA测序（RNA-seq）技术分析了β-2属HPV（38b、107及122型）E6/E7蛋白介导的差异表达基因。本研究揭示了β-2属HPV基因型同样具备的细胞转化潜能，其中尤以HPV122型为著。此类β-2型HPV可调控部分已知由α-HPV 16及18型调控的基因。

实验整体设计：从感染携带HPV 16、18、38b、107及122型E6/E7基因的病毒颗粒的成纤维细胞中提取总RNA，每组设置3次生物学重复。鉴于HPV16及18型已被证实具有细胞转化特性，将其作为阳性对照；以转导空pLVX载体的原代成纤维细胞作为基础对照，所有细胞模型均与该对照进行比较。为获取基因的差异表达情况，将6份FB-pLVX重复样本分别与每种细胞模型的3份重复样本进行比对分析。