Previous research identified the expression of EDN3 in granulosa cells of preovulatory follicles in chickens. Notably, the expression level of EDN3 in Silky Fowl with low egg-laying performance was significantly higher than that in high-yield laying breed White Leghorn. Given the crucial role of granulosa cells in follicular development and maturation, it is very important to study the effect of EDN3 on the biological function of granular cells. In this study, an EDN3 overexpression plasmid was constructed and transfected into granular cells. The viability of these cells was detected using quantiative (qPCR), Cell Counting Kit-8 (CCK8), and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Gonadal hormone synthesis was detected using enzyme-linked immunosorbent assay (ELISA) techniques. Finally, transcriptome sequencing was employed to identify differentially expressed genes. Result showed thatoverexpression of EDN3 was observed to promote cell viability. In addition, it significantly inhibits the expressions of LHR and cAMP-PKA signaling pathways. Cell transcriptome sequencing data displayed that EDN3 can upregulate energy metabolism and immune-related signaling pathways, whereas follicle maturation and the GnRH signaling pathway were downregulated. In conclusion, this study demonstrates that EDN3 can enhance granulosa cell viability and inhibit the expression of LHCGR, a process likely mediated through the cAMP-PKA signaling pathway. However, further evidence is required to substantiate the regulatory relationship between EDN3 and the cAMP-PKA signaling pathway.

Heme oxygenase-1 (HO-1), encoded by HMOX1, catalyzes the rate-limiting step of heme degradation and generates biliverdin, carbon monoxide, and ferrous iron, thereby linking heme turnover with redox regulation and stress-associated signaling. In birds, biliverdin is retained as a major heme-derived product, but the cellular consequences of HMOX1 perturbation remain insufficiently defined. Here, CRISPR/Cas9-mediated editing was used to generate a heterogeneous HMOX1-edited population in Chicken hepatocellular carcinoma-derived cells. The selected sgRNA reduced HO-1 protein abundance by approximately 47%, and no detectable cleavage was observed at the seven predicted high-risk off-target loci examined. Compared with vector-control cells, HMOX1-edited cells exhibited intracellular heme accumulation, reduced biliverdin levels, increased oxidation-sensitive fluorescence, and reduced CCK-8 absorbance values, indicating disruption of heme–biliverdin metabolic and redox homeostasis. RNA sequencing identified 2650 differentially expressed genes, including 951 upregulated and 1699 downregulated genes. Downregulated genes were mainly enriched in immune, cytokine, MAPK/stress, and extracellular signaling-associated pathways, whereas DNA replication and cell-cycle-related genes were increased. Enrichment-term association and STRING functional-association analyses further identified a coordinated module involving IL1B, JUN, NFKBIA, IRF1, TGFB1, IL10, CCL5, and PTGS2. Independent RT-qPCR analysis confirmed selected expression trends. These findings show that heterogeneous HMOX1 editing and reduced HO-1 abundance are associated with disruption of the avian heme–biliverdin metabolic axis and coordinated remodeling of basal immune, stress, extracellular signaling, and cell-cycle-associated transcriptional programs in Chicken hepatocellular carcinoma-derived cells.