The Induced Expression of BPV E4 Gene in Equine Adult Dermal Fibroblast Cells as a Potential Model of Skin Sarcoid-like Neoplasia

Abstract

The equine sarcoid is one of the most common neoplasias in the Equidae family. Despite the association of this tumor with the presence of bovine papillomavirus (BPV), the molecular mechanism of this lesion has not been fully understood. The transgenization of equine adult cutaneous fibroblast cells (ACFCs) was accomplished by nucleofection, followed by detection of molecular modifications using high-throughput NGS transcriptome sequencing. The results of the present study confirm that BPV-E4- and BPV-E1^E4-mediated nucleofection strategy significantly affected the transcriptomic alterations, leading to sarcoid-like neoplastic transformation of equine ACFCs. Furthermore, the results of the current investigation might contribute to the creation of in vitro biomedical models suitable for estimating the fates of molecular dedifferentiability and the epigenomic reprogrammability of BPV-E4 and BPV-E4^E1 transgenic equine ACFC-derived sarcoid-like cell nuclei in equine somatic cell-cloned embryos. Additionally, these in vitro models seem to be reliable for thoroughly recognizing molecular mechanisms that underlie not only oncogenic alterations in transcriptomic signatures, but also the etiopathogenesis of epidermal and dermal sarcoid-dependent neoplastic transformations in horses and other equids. For those reasons, the aforementioned transgenic models might be useful for devising clinical treatments in horses afflicted with sarcoid-related neoplasia of cutaneous and subcutaneous tissues.

Assessment of BPV-1 Mediated Matrix Metalloproteinase Genes Deregulation in the In Vivo and In Vitro Models Designed to Explore Molecular Nature of Equine Sarcoids

“…The high-throughput analysis of transcriptomic signatures [9] was used to identify the MMP genes for further analyses based on applying cDNA microarrays to compare the gene expression patterns in healthy skin and sarcoid tissue samples. Additionally, using the RNA-seq technique, a panel of studies was accomplished to estimate the MMP gene expression profiles in both BPV-E4ˆE1 transfected ACFC lines and their counterparts transfected with empty vectors as a negative control group [10]. Considering both data sets generated (designated as GSE83430 and GSE193906), genes that belonged to the MMP family and their corresponding fold change values were achieved.…”

OCT6 inhibits differentiation of porcine-induced pluripotent stem cells through MAPK and PI3K signaling regulation

“…Highly reprogrammable NDCs may exhibit an enhanced capacity to sustainably perpetuate stemness- and pluripotency-related molecular attributes. The latter, in turn, could enhance the epigenetic dedifferentiation capacity of OE-OCT6 iPSCs in SCNT-derived embryos, conceptuses, and progenies generated for transgenic and biomedical research, not only for pig-to-human xenotransplantation and tissue engineering, but also for regenerative and reconstructive surgical treatments in humans and other mammalian species ( Berthelsen et al, 2021 ; Podstawski et al, 2022 ; Song et al, 2016 ; Wiater et al, 2021a , 2021b ; Xu et al, 2022 ; Yu et al, 2021 ; Zhao et al, 2020 ).…”

Tracking the Molecular Scenarios for Tumorigenic Remodeling of Extracellular Matrix Based on Gene Expression Profiling in Equine Skin Neoplasia Models

“…The procedures encompassing the ex vivo establishment and nucleofection of ACFC lines were comprehensively described in the study by Podstawski et al [ 52 ]. Briefly, the primary cultures of horse skin-derived fibroblast cells followed by mitotically stable ACFC lines were established in compliance with the method developed and optimized by Tomasek et al [ 53 ].…”

“…As thoroughly specified in our previous investigation [ 52 ], immediately after ACFC lines had reached 90% confluence, they were trypsinized, centrifuged in Tissue Culture Medium 199 (TCM 199; Sigma-Aldrich, Merck Life Sciences, Poznań, Poland), supplemented with 5% FBS (Gibco, Thermo Scientific, Waltham, MA, USA) and then subjected to in vitro transgenization by nucleofection using the T-REx system (Invitrogen, Thermo Scientific, Waltham, MA, USA) and BPV1-E1^E4 gene construct. The cell transfection was performed with the aid of the Amaxa Nucleofector™ II Device (Amaxa Biosystems, Lonza, Medianus, Kraków, Poland) and by using a dedicated reagent kit as follows: Amaxa™ Normal Human Dermal Fibroblast-Adult (NHDF-Adult) and Nucleofector™ Kit (Lonza, CELLLAB, Warsaw, Poland).…”