TRPS1 Affects The Transition Of Mouse 2-cell Embryos To 4-cell Embryos Through Akt/Creb Signaling Pathway

Objective:To explore Trps1 participates in the development of mouse 2-cell embryos to 4-cell embryos by affecting the Akt/Creb signaling pathway.To clarify the specific mechanism that affects AKT kinase activity by Trps1,and provide new ideas for overcoming the developmental block of in vitro fertilized eggs.Methods:1.1-cell embryos were collected 18 h after h CG,cultured in KSOM medium for 3h,and1-cell embryos with female and male pronuclei were collected,and divided into blank control group(KSOM culture group),negative control group(negative si RNA),Trps1-si RNA injection group,and AKT agonist complement group(Trps1-si RNA+4μg/m L SC-79),these 1-cell embryos were cultured in KSOM medium.(1)To observe the subsequent development of 1-cell embryos in each group and count the development rate;(2)Real-Time PCR was used to detect the changes of Trps1,Akt and Creb m RNA expression in each group of 2-cell embryos;(3)Immunofluorescence staining and laser confocal scanning microscope were used to observe the changes of TRPS1,AKT,p-AKT,CREB,and p-CREB protein expression in each group of 2-cell embryos.2.Cultivation and treatment of ovarian cancer cell line S-KO-V3: The S-KO-V3 cells were taken out from the liquid nitrogen tank and resuscitated and passed down.When the confluence is nearly to 70%,the S-KO-V3 cells were divided into negative control group(negative si RNA),TRPS1-si RNA transfection group,and AKT agonist complement group(TRPS1-si RNA+4μg/m L SC-79).(1)After 24 h of culture,the total RNA of the cells was extracted,and the expression of TRPS1,AKT,CREB m RNA in each group was detected by Real-Time PCR;(2)After 48 h of culture,the total cell protein was extracted,and Western blot and immunofluorescence staining were used to detect the changes of expression and location of AKT,p-AKT,CREB,and p-CREB proteins in each group.3.Ch IP-seq experiment and verification: S-KO-V3 cells were collected.Ch IP-seq detection and the KEGG pathway enrichment method were used to screen out the signal pathways closely related to CREB,and then to search for the downstream genes combined with TRPS1 tightly,which werevalidated in S-KO-V3 cell line and preimplantation embryos.Results:1.The results of mouse preimplantation embryo culture showed that after 1-cell embryos were injected with Trps1-si RNA,the transition rate of 2-cell embryos to 4-cell embryos was significantly reduced(P<0.01).Compared to Trps1-si RNA injection group,the transition rate of2-cell embryos to 4-cell embryos in the AKT agonist complement group was significantly increased(P<0.01).2.Real-time PCR results showed that the injection of Trps1-si RNA can significantly reduce the expression of Trps1 m RNA in 2-cell embryos(P<0.001),but the expression of Akt and Creb m RNA did not change significantly(P>0.05).Immunofluorescence staining results showed that the injection of Trps1-si RNA can reduce the expression of TRPS1 protein in 2-cell embryos(P<0.001).It also reduce the expression of p-AKT and p-CREB proteins(P<0.001),but the expression of AKT and CREB proteins did not change significantly(P>0.05).After the treatment of AKT agonist,p-AKT and p-CREB proteins expression increased significantly(P<0.001).3.Real-time PCR results showed that after TRPS1-si RNA transfection,the expression of TRPS1 m RNA in S-KO-V3 cells was down-regulated,but the expression of AKT and CREB m RNA did not change significantly(P>0.05).Compared to the TRPS1-si RNA transfection group,the expression of AKT m RNA in the AKT agonist complement group increased significantly(P<0.001),and the expression of CREB m RNA also increased(P<0.01).Western blot and immunofluorescence staining showed that with the down-regulation of TRPS1 protein,p-AKT and p-CREB proteins were also decreased,while AKT and CREB proteins remained unchanged.After the treatment of AKT agonist,AKT,p-AKT and p-CREB proteins expression increased significantly.4.Applying S-KO-V3 cell Ch IP-seq results and combining with KEGG pathway enrichment analysis,It is found that the PI3K/Akt signaling pathway is closely related to CREB.The downstream gene PDE4 D,which has multiple binding domains with TRPS1,is also screened out.Real-Time PCR and Western blot detection showed that in S-KO-V3 cells,as the expression of TRPS1 decreased,the expression level of PDE4 D also decreased.Immunofluorescence staining showed that in preimplantation embryos of mice,injection of Trps1-si RNA can reduce the expression level of PDE4 D protein in 2-cell embryos.Conclusion:In summary,Trps1 may affect the activity of AKT kinase by regulating the transcription process of Pde4 d and participate in the development of mouse 2-cell embryos to 4-cell embryos through the Akt/Creb signaling pathway.