Effects of FOXP3?2?7 exogenous expression on gene expression in a non-tumorigenic keratinocyte model

Cervical cancer (CC) is the fourth most common type of cancer among women. FOXP3, a critical transcription factor involved in the biology of regulatory T cells, has been detected as highly expressed in the tumor cells of CC patients. However, its biological role in CC, particularly in the keratinocytes, remained unclarified. Therefore, this work aimed to uncover the effects of FOXP3 on the biology of the tumoral cells. For this purpose, a model with constitutively expression of FOXP3?2?7was established to evaluate its role in proliferation, migration, and cell division. As well, RNAseq was performed to identify differentially expressed genes and enriched pathways modulated by FOXP3?2. The exogenous expression of FOXP3?2?7 promotes cell division, proliferation, and migration. The transcriptomic analyses highlight the upregulation of multiple genes with protumor activities. Moreover, immunological and oncogenic pathways were detected as highly enriched. These data support that FOXP3?2?7 in epithelial cells induces cancer-related hallmarks and provides information about the molecular events triggered by this isoform, which could be important for developing CC. Overall design: Total RNA extraction was performed by duplicate from cell models obtained from HaCaT transduced with viral particles containing the FOXP3?2?7 gene, HaCaT cells transduced with the empty vector were used as controls. RNA-seq was performed to obtain the differentially expressed genes and the enriched pathways modulated by FOXP3?2?7. Next-generation RNA sequencing with Nova Seq 6000 Illumina platform was conducted by Novogen Bioinformatics Technology Co., Ltd (Beijing, China); independent sample duplicates of total RNA from HaCaT-LVX and HaCaT-FOXP3?2?7 were sent preserved in RNAstable (Cat. No. 93221-001, Biomatrica, San Diego, CA). Retrieved data was analyzed in both open-source platforms, RStudio (Version 1.4.1717) and Galaxy (Version 22.05.1) (https://www.usegalaxy.org). The sequencing quality of the returned data (FASTQ archives) was analyzed with FASTQC (Version 0.11.9). Subsequently, alignment to the Homo sapiens genome (Version 42) (GRCh38.p13) was performed with Subjunc aligner (Version 2.0.0) from the Rsubread package (Version 3.14). Afterwards, BAM files were processed with featureCounts (Version 2.0.1). Normalization of reads counts as FPKM (Frag-ments per Kilobase of exon per Million Mapped Fragments) was achieved using DESeq2 tool (Version 1.36.0).