Transcriptome profiling reveals transcriptional regulation of Protegrin-1 on immune defense and development in porcine granulosa cells

Abstract

Protegrin-1 (PG1) is an antimicrobial peptide (AMP) that has garnered increasing attention due to its potent immune defense activity. Our previous studies demonstrated the ability of PG1 to enhance proliferation and inhibit apoptosis of porcine granulosa cells (GCs) under oxidative stress. GCs play a crucial role in ovary follicular development. However, the specific function and underlying mechanisms of AMP in follicular development still need further elucidation. The present study aimed to comprehensively explore the biological effects of PG1 on porcine GCs using transcriptome profiling by RNA sequencing technology. Isolated GCs were incubated with or without PG1 for 24 h and transcriptome-wide analysis was exerted to identify differentially expressed genes (DEGs). The results of expression analysis revealed 1,235 DEGs, including 242 up-regulated genes and 993 down-regulated genes (|log2 (FoldChange)| > 1; adjusted P-value < 0.05). The expression levels of 7 selected DEGs were validated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis, which was consistent with the RNA-sequencing data. Among the significant DEGs, several genes associated with GC function and ovarian follicle development were identified, such as estrogen receptor 2 (ESR2), growth and differentiation factor 6 (GDF6), cell division cycle 20 homolog (CDC20), Notch3, ephrin and Eph receptor system, Egl nine homolog 3 (EGLN3), and BCL2 like 14 (BCL2L14). Gene Ontology (GO) analysis revealed that the top three significant GO terms were inflammatory response, defense response, and granulocyte migration. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis presented that DEGs were mainly enriched in the immune system, infectious disease, signaling molecules and interaction, and immune disease. Furthermore, Ingenuity Pathway Analysis (IPA) predicted that the top activated pathway was Liver X Receptor (LXR)/ Retinoid X Receptor (RXR) Activation which is known to be associated with female reproduction. Predicted protein–protein interactions (PPIs) analysis identified complement C3 (C3) as the top node with the highest degree of network connection and revealed that DEGs in the sub-networks were involved in cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, chemokine signaling pathway, and metabolic process. In conclusion, this study expanded the understanding of the effects of PG1 on porcine GCs at the transcriptomic level and provided a theoretical basis for further investigation into the role of PG1 in immune defense and mammalian ovarian follicular development.