Objective To compare the differences in transcriptional responses between wild-type and eight-gene editing (8GE) porcine endothelial cells stimulated by human serum.

Methods Wild-type porcine endothelial cells and 8GE porcine endothelial cells were used to establish the wild-type unstimulated group (WT-Ctrl), wild-type human-serum-stimulated group (WT-HS), 8GE unstimulated group (8GE-Ctrl) and 8GE human-serum-stimulated group (8GE-HS). RNA-sequencing was performed to detect transcriptional alterations in each group. Differentially expressed gene (DEG) analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, as well as protein-protein interaction (PPI) network analysis were adopted for transcriptional profiling.

Results Good reproducibility was observed among samples from the four groups, and the global expression profiles were distinctly separated across treatment groups. A total of 543 differentially expressed genes were identified in the WT-HS group compared with the WT-Ctrl group. These genes were mainly enriched in inflammatory and innate-immunity-related pathways, including immune response, chemotaxis, interleukin (IL) -17, Toll-like receptor, mitogen-activated protein kinase (MAPK), tumor necrosis factor (TNF), and cytokine-receptor interaction pathways. Candidate hub-node genes screened from the PPI network included IL6, CCL2, CXCR4, NFKBIA and FOS. Compared with the WT-Ctrl group, 1 967 differentially expressed genes were screened in the 8GE-Ctrl group, which were involved in processes such as immune regulation and extracellular matrix organization. A total of 1 912 differentially expressed genes were identified in the 8GE-HS group relative to the 8GE-Ctrl group, with candidate hub-node genes from the PPI network mainly comprising TLR4, NFKBIA, TRAF2 and TRAF3. Furthermore, 3 054 differentially expressed genes were found between the 8GE-HS group and the WT-HS group, which were predominantly enriched in the PI3K-Akt, MAPK, extracellular-matrix-receptor interaction, cell adhesion molecule, regulation of actin cytoskeleton, and focal adhesion pathways.

Conclusions Human serum stimulation may trigger a xenogeneic-rejection-like transcriptional response in wild-type porcine endothelial cells, characterized by elevated expression of inflammatory-factor-related genes, enhanced chemokine signaling and activated innate immunity. 8GE endothelial cells still maintain transcriptional responsiveness to human serum stimulation. Nevertheless, their response pattern shifts from output of inflammatory-chemotactic effects toward upstream immune-signal regulation.