PPP2R2C Mediates MVP Regulation Of JAK/STAT1 Pathway And PTEN Nuclear Translocation To Repair Immune Premature Ovarian Insufficiency

BackgroundPremature ovarian insufficiency(POI)is a common endocrine disorder affecting female reproductive health,and clinical treatments for POI are limited in their availability and effectiveness.Autoimmune disorders are an important mechanism of autoimmune POI.The induction of follicular atresia by granulocytes acting on immune cells such as cytotoxic T cells is a important aspect of autoimmune POI,but the immune regulatory mechanisms at the granulocyte level have not yet been elucidated,hindering the development of immunotherapy for POI.ObjectiveIn this study,we identified PPP2R2C as a candidate target gene for autoimmune POI based on the results of second-generation sequencing and bioinformatics analysis of ovarian tissues from mice with autoimmune POI induced by zona pellucida 3 peptide(pZP3).The study was conducted to explore the feasibility of PPP2R2C as a therapeutic target for POI,and to provide a theoretical basis for exploring the pathogenesis of autoimmune POI and new therapies.Methods1.The expression of PPP2R2C in ovarian tissues of autoimmune POI mice was detected by fluorescent quantitative PCR(qRT-PCR)and immunohistochemical staining.An IFN-y-induced immunoinflammatory granulosa cell model was established to simulate the immune damage state of ovarian granulosa cells in vitro.The effects of PPP2R2C on granulosa cell proliferation,apoptosis,cycling,hormone synthesis and PP2A phosphatase activity were verified by CCK-8,flow cytometry,qRT-PCR and enzyme-linked immunosorbent assay(ELISA)in this model.2.Protein immunoprecipitation(Co-IP)combined with mass spectrometry was used to find the target protein of PPP2R2C,to clarify the interaction between the target protein MVP and PPP2R2C,and to construct truncated segments to find their binding structural domains.Flow cytometry and ELISA were used to clarify that MVP regulates apoptosis and E2 levels in granulosa cells.Response experiments demonstrated that PPP2R2C overexpression mediated the reduction of granulosa cell apoptosis and synthesis of E2 by MVP.3.KEGG,Reactome,GSEA analysis of RNA-seq data from ovaries of POI mice clarified the key links of immune abnormalities.qRT-PCR,western blot demonstrated that IFN-y activates the JAK/STAT1-MHC Ⅰ/Ⅱ signaling axis.qRT-PCR,western blot clarified that knockdown of MVP promotes Co-IP,immunofluorescence(IF)assay verified that MVP interacts with P-STAT1 and participates in P-STAT1 nuclear translocation.4.The PPP2R2C and MVP regulated the repair of DNA damage in granulosa cells by western blot and IF.Co-IP,western blot and IF demonstrated that MVP acted directly on PTEN and regulated its expression and nuclear translocation.5.The ovarian structure,motility cycle,follicle number at each level,serum E2,FSH and AMH levels,ovarian granulosa cell apoptosis,ovarian tissue ki67 expression,ovarian tissue mRNA expression of MHC I-related genes and serum IFN-γ,IL-1β and other inflammatory factors levels were compared to verify the ovarian structure,endocrine and immune functions of ovaries of mice with autoimmune POI induced by in situ injection of PPP2R2C overexpressing lentivirus to repair pZP3.Results1.PPP2R2C expression was elevated in the ovaries of autoimmune POI mice and in granulosa cells with IFN-γ-induced immune inflammatory injury.Overexpression of PPP2R2C repaired the proliferation,cycling and hormone synthesis functions of granulosa cells with IFN-y-induced injury and reduced apoptosis.2.PPP2R2C interacts with an intermediate structural domain consisting of amino acid residues(310-620)of MVP and mediates the reduction of IFN-y-induced apoptosis by MVP in granulosa cells with IFN-y-induced immune inflammation.3.IFN-y induced activation of the JAK/STAT1 pathway and increased MHC I/II expression in granulosa cells.MVP mediates STAT1 phosphorylation and interacts with it to promote P-STAT1 nuclear translocation and MHC I expression.4.PPP2R2C and MVP promote IFN-y-induced DNA damage repair in immune injury granulosa cells.MVP interacts with PTEN to promote PTEN nuclear translocation and is associated with granulosa cell DNA damage mechanisms.5.Ovarian in situ injection of lentivirus overexpressing PPP2R2C promoted the recovery of ovarian structure,endocrine and immune function in autoimmune POI mice.ConclusionPPP2R2C mediates the MVP-regulated JAK/STAT1 signaling pathway and PTEN nuclear translocation to affect the expression of the granulosa cell antigen-presenting molecule MHC I and DNA damage repair,respectively,improving ovarian granulosa cell immune damage and promoting the repair of autoimmune POI.