Abstract:Objective Utilizing human ovarian granulosa tumor cells as the cellular model, we tried to investigate the effect of roofplate-specific spondin 2 (RSPO2) on the oxidative stress and apoptosis level of ovarian granulosa cells. Methods We performed transient transfection of RSPO2 overexpression plasmid and small interfering RNA (siRNA) in COV434 cells. Real-time Quantitative PCR (RT-PCR) and Western blot were used to detect efficiency of overexpression and interference. The reactive oxygen species assay kit was used to determine the amount of intracellular reactive oxygen species. The apoptosis level was detected by flow cytometry. RT-PCR and Western blot were used to determine the expression levels of oxidative stress and apoptosis-related genes. Co-immunoprecipitation was used to detect interacting proteins. Results The overexpression and interference of RSPO2 were successfully realized. Overexpression of RSPO2 significantly inhibited the level of reactive oxygen species in COV434 cells (P < 0.05) and extremely significant inhibited the early apoptosis of granulosa cells (P < 0.01), interference of RSPO2 obtained the opposite results. Overexpression of RSPO2 significantly upregulated the mRNA and protein levels of SOD1, SOD2, CAT (P < 0.05), and significant downregulated the levels of caspase 3 and caspase 8 (P < 0.05). On the contrary, interference of RSPO2 significantly downregulated the levels of SOD1 and CAT (P < 0.05), and significantly upregulated the level of caspase 3 and caspase 8 (P < 0.05). We found the protein interactions between RSPO2, SOD1, CAT and caspase 3. Conclusion Interfering with RSPO2 promotes the accumulation of reactive oxygen species within cells, downregulates the expression of SOD1 and CAT, thereby enhancing oxidative stress. Additionally, interfering with RSPO2 upregulates the expression of caspase 3 and caspase 8, promoting cell apoptosis. These findings suggest that RSPO2 plays a crucial role in ovarian granulosa cells.