The Molecular Mechanism Of NF2/SWH Pathway Regulating Granulosa Cell Proliferation,Differentiation And Apoptosis Of The Ovarian Prehierarchical Follicles In Hen

NF2(neurofibromin 2)molecules inhibits tumorigenesis,regulates embryonic development by regulating cell growth and development in mammals and Drosophila.SWH pathway(also known as Hippo pathway)plays an important regulatory role in the growth and development of chicken follicles,regulating follicular development by inhibiting granulosa cell proliferation and promoting granulosa cell apoptosis.However,the role and molecular mechanism of NF2 in the proliferation,differentiation,and apoptosis of chicken prehierarchical granulosa cells through the SWH signaling pathway are currently unclear.This study,Hylan brown laying hens were used as test materials to study by using RT-qPCR,Western blot,Elisa,gene knockdown,gene over-expression,flow cytometry and other technical techniques to detect and analyze the spatiotemporal expression characteristics of NF2 gene and protein in chicken follicles and granulosa cells,revealing the regulatory role of NF2 molecules on the proliferation,differentiation,and apoptosis of chicken follicular granulosa cells;Further proving the molecular biological effects of SWH signaling pathway mediated NF2 regulation;We have thoroughly analyzed the molecular mechanism by which the NF2/SWH signaling pathway regulates the proliferation,differentiation,and apoptosis of chicken prehierarchical follicular granulosa cells through the downstream effector factor YAP1-p38 MAPK signaling pathway.The main research work and results carried out are as follows:1.The spatiotemporal expression characteristics of chicken NF2 molecule in prehierarchical follicles and granulosa cellsThe spatiotemporal expression pattern of NF2 in chicken prehierarchical follicles and granulosa cells was measured and analyzed by RT-qPCR and immunofluorescence and other technologies from the transcription and protein level.The results showed that NF2 gene m RNA was expressed in prehierarchical follicles and granulosa cells at different stages of development;NF2 protein was mainly expressed in prehierarchical follicles and granulosa cells.Further analysis with laser confocal microscopy showed that NF2 protein was expressed in the cytoplasm of granulosa cells.This result clarified the spatiotemporal expression characteristics of chicken NF2,laying the foundation for further revealing its function and molecular mechanism of action in follicular development.2.The role of NF2 in regulating the proliferation,differentiation and apoptosis of chicken prehierarchical follicular granulosa cellsTargeted interference,EDU and flow cytometry were used to detect the roles of NF2 on the proliferation,differentiation and apoptosis of chicken prehierarchical follicular granulosa cells at m RNA,protein and cell levels.Firstly,construct the NF2 gene interference vector.After the constructed NF2 gene interference vector is transfected into chicken prehierarchical follicular granulosa cells,detect the changes of FSHR,MYC,GDF9,STAR,CYP11A1,BCL2 and CASP3 genes at the level of m RNA and protein,and then detect the role of NF2 on the proliferation,differentiation and apoptosis of chicken prehierarchical follicular granulosa cells at the cell level using EDU and flow cytometry.The results showed that NF2 shRNA significantly promoted the proliferation and differentiation of prehierarchical granulosa cells(P<0.05)and inhibited the apoptosis of prehierarchical granulosa cells(P<0.05).NF2 shRNA significantly up-regulated the m RNA and protein expression of FSHR,MYC,GDF9,STAR,CYP11A1 and BCL2(P<0.05),and significantly down-regulated the m RNA and protein expression of CASP3(P<0.05).The results showed that NF2 may inhibit the proliferation and differentiation of prehierarchical granulosa cells,promote GC apoptosis and regulate the growth and development of prehierarchical follicles in chicken by regulating the biomarker genes to follicular selection,GC proliferation,differentiation and apoptosis.3.The molecular biological effects of SWH signaling pathway mediated NF2regulationThe role of NF2 shRNA on the expression of STK3,STK4,LATS1,LATS2 and YAP1,the key members of SWH signaling pathway,was detected at the m RNA and protein levels by RT-qPCR,Western blot and other techniques.After adding STK3/4 inhibitor(XMU-MP-1),the role of NF2 shRNA on the expression of STK3,STK4,LATS1,LATS2 and YAP1 was detected.At the same time,EDU and flow cytometry were used to detect the role of NF2 shRNA on the proliferation,differentiation and apoptosis of chicken prehierarchical follicular granulosa cells at the cell level.The results showed that: 1)10 μ M XMU-MP-1 significantly inhibited the expression of STK3,STK4,LATS1,LATS2 m RNA and phosphorylated protein(P<0.01),and significantly promoted the expression of YAP1 m RNA and phosphorylated protein(P<0.01);2)NF2 shRNA significantly decreased the expression of STK3,STK4,LATS1,LATS2 m RNA and phosphorylated protein(P<0.05),and significantly increased the expression of YAP1 m RNA and phosphorylated protein(P<0.05).At the same time,NF2 shRNA significantly promoted the proliferation and differentiation of prehierarchical granulosa cells(P<0.05),and significantly inhibited GC apoptosis(P<0.05);3)Compared with NF2 shRNA group,XMU-MP-1+NF2 shRNA group inhibited the expression of STK3 and STK4 after inhibiting STK3/4(P<0.05);after add XMU-MP-1,the effect of NF2 shRNA on LATS1,LATS2 and YAP1 was reversed(P<0.05)and on the proliferation,differentiation and apoptosis of prehierarchical granulosa cells was reversed(P<0.05).Further clarify that SWH signaling pathway mediates NF2 to inhibit the proliferation and differentiation of prehierarchical granulosa cells and promote the granulosa cells apoptosis.4.Molecular mechanism of of NF2/SWH signaling pathway regulating the GC proliferation,differentiation,and apoptosisRT-qPCR,ELISA,Western Blot,flow cytometry and other methods were used to detect the roles of co-transfection NF2 siRNA and YAP1 siRNA on the proliferation,differentiation,apoptosis,p38 MAPK and mitochondrial function-related indicators of granulosa cells.The results showed that after co-transfection of NF2 siRNA and YAP1 siRNA,the effect of NF2 siRNA on cell proliferation and apoptosis was reversed(P<0.05).The effect of NF2 siRNA on FSHR,GDF9,MYC,STAR,CYP11A1,BCL2 and CASP3 m RNA expression was reversed(P<0.05).NF2 siRNA significantly decreased ROS content and cytochrome C release(P<0.05),significantly increased mitochondrial membrane potential,ATP content and mitochondrial COI gene copy number(P<0.05).After co-transfection of NF2 siRNA and YAP1 siRNA,the effect of NF2 siRNA on mitochondrial function was reversed(P<0.05).It is proved that NF2/SWH inhibits the proliferation and differentiation of granulosa cells and promote GC apoptosis and affects the mitochondrial function through the downstream effector factor YAP1 of SWH signaling.NF2 siRNA significantly decreased the expression of p-p38 MAPK protein(P<0.05),and YAP1 siRNA significantly increased the expression of p-p38 MAPK protein(P<0.05).After co-transfection of NF2 siRNA and YAP1 siRNA,the effect of NF2 siRNA on p-p38 MAPK was reversed(P<0.05).This indicates that NF2/SWH negatively regulates the p38 MAPK pathway through the downstream effect factor YAP1 of SWH.In order to further explore that NF2/SWH signaling pathway regulates mitochondrial function through p38 MAPK pathway and regulates apoptosis of granulosa cells.In this experiment,U46619(p38MAPK activator)and SB203580(p38 MAPK inhibitor)were added for detection.The results showed that compared with NF2 siRNA,NF2 siRNA transfected with U46619 after2 h pretreatment significantly increased ROS content and cytochrome C release(P<0.05);ATP content,mitochondrial membrane potential and mitochondrial COI gene copy number were significantly decreased(P<0.05);significantly inhibit the expression of BCL2 gene m RNA and protein(P<0.05)and promote the expression of CASP3 gene m RNA and its protein,as well as the rate of cell apoptosis(P<0.05).The effects of NF2 siRNA transfected with SB203580 on ROS content,cytochrome C release,ATP content,mitochondrial membrane potential and mitochondrial COI gene copy number were reversed after 2 h pretreatment with U46619(P<0.05);The effect on BCL2,CASP3 gene expression and GC apoptosis was reversed(P<0.05).It proved that NF2/SWH signaling pathway negatively regulates mitochondrial function through the downstream effector factor YAP1-p38 MAPK pathway,thereby promoting apoptosis of granulosa cells.This study revealed that NF2 molecules have the effect of inhibiting the proliferation and differentiation of prehierarchical follicular granulosa cells,promoting apoptosis of prehierarchical follicular granulosa cells,and deeply analyzed the molecular mechanism by which NF2 molecules regulate the proliferation,differentiation,and apoptosis of prehierarchical follicular granulosa cells through the SWH signaling pathway.