Effect Of PITX2 On E₂/P₄ Secretion And Proliferation Of Ovarian Granulosa Cells In Dairy Goats

Paired like homeodomain 2（PITX2）is a member of the PITX family of pituitary homeodomains.Studies have shown that PITX2 can directly or specifically activate typical WNT signaling pathways through WNT ligands,and WNT signaling can regulate ovarian steroidogenesis and cell proliferation.At present,the effect of PITX2 on the secretion of E₂,P₄ and proliferation of ovarian granulosa cells in dairy goats is not yet known.This study takes ovarian granulosa cells of dairy goat as the research object.Through the construction of PITX2 overexpression vector and the synthesis of its interfering RNA,exploring the regulation of PITX2 on the secretion of E₂,P₄ and proliferation in granulosa cells,and clarifying the biological role of PITX2 in follicular development and ovulation.The main results are as follows:1.Construction of PITX2 overexpression vector and synthesis of PITX2-siRNAPITX2 is expressed in 11 tissues including goat ovary,kidney,heart,liver,breast,uterus,spleen,lipid,muscle,oviduct and lung.Among them,the expression of uterus is the highest,and it is also higher in lung,oviduct,kidney and liver.The expression of ovary,heart,muscle and lipid is less.The full-length sequence of the CDS region of the PITX2gene amplified by PCR was ligated to the pcDNA3.1 vector and named pcDNA-PITX2.At the same time,PITX2 interfering RNA was synthesized and named PITX2-siRNA.RT-qPCR and Western blot were used to detect PITX2 overexpression and interference efficiency.The results showed that PITX2 overexpression significantly upregulated the expression of PITX2 in granulosa cells;PITX2-siRNA significantly down-regulated the expression of PITX2 in granulosa cells.2.The regulation of PITX2 on the secretion of E₂,P₄ in ovarian granulosa cells of dairy goatPITX2 overexpression and PITX2-siRNA were transfected into granulosa cells respectively.ELISA results showed that overexpression of PITX2 significantly up-regulated the secretion of E₂,P₄ in granulosa cells,and knocking down PITX2 significantly down-regulated the secretion of E₂,P₄ in granulosa cells;further,RT-qPCR and Western blot were used to detect the effect of PITX2 on the gene expression of key enzymes in steroidogenesis.The results showed that PITX2 overexpression significantly up-regulated the expression of CYP11A1,CYP19A1 and 3β-HSD,and PITX2-siRNA significantly down-regulated the expression of CYP11A1,STAR,CYP19A1 and 3β-HSD.The above results indicate that PITX2 regulates the secretion of E₂,P₄ by regulating the gene expression of key enzymes in steroidogenesis.3.PITX2 regulates the secretion of E₂,P₄ in ovarian granulosa cells of dairy goat through WNT/β-catenin signaling pathwayTo investigate whether PITX2 regulates the secretion of E₂,P₄ by activating the WNT/β-catenin signaling pathway,granulosa cells were treated with Salinomycin sodium salt（WNT/β-catenin signaling pathway inhibitor）at a final concentration of 0.5 m M for 24hours and then transfected with PITX2 overexpression,ELISA results showed that the Salinomycin sodium salt treatment group significantly down-regulated the secretion of E₂,P₄compared with the control group,while overexpression of PITX2 significantly reduced the inhibitory effect of Salinomycin sodium salt on the secretion of E₂,P₄ in granulosa cells.Further research results showed that overexpression of PITX2 significantly up-regulated the expression of DVL1 andβ-catenin,and PITX2-siRNA significantly down-regulated the expression of DVL1 andβ-catenin.4.PITX2 affects the secretion of E₂,P₄ by regulating the expression of CTNNB1In order to study the effect of CTNNB1 on PITX2 regulating the secretion of E₂,P₄,CTNNB1-siRNA was synthesized,RT-qPCR and Western blot were used to verify its interference efficiency.The results showed that CTNNB1-siRNA significantly down-regulated the expression of CTNNB1 in granulosa cells.Further,Western blot was used to detect the effect of CTNNB1-siRNA on the gene expression of key enzymes in steroidogenesis.The results showed that knocking down CTNNB1 significantly down-regulated the expression of the protein levels of CYP11A1,STAR,CYP19A1 and3β-HSD.Co-transfection of pcDNA-PITX2 and CTNNB1-siRNA,showed that the pcDNA3.1+CTNNB1-siRNA group significantly down-regulated the protein levels of CYP11A1,STAR,CYP19A1 and 3β-HSD,while the co-treatment group compared with The pcDNA3.1+CTNNB1-siRNA group significantly increased the expression of the protein levels of CYP11A1,STAR,CYP19A1 and 3β-HSD.The above results indicate that PITX2regulates the secretion of E₂,P₄ by regulating the expression of CTNNB1 and affecting the gene expression of the key enzymes in steroidogenesis.5.PITX2 promotes the proliferation of granulosa cellCCK-8 detects the effect of PITX2 on viability in granulosa cell and Ed U detects the effect of PITX2 on the proliferation of granulosa cell.The results show that overexpression of PITX2 significantly increases the viability and the proliferation rate of granulosa cells,while PITX2-siRNA significantly reduces the viability and the proliferation rate of granulosa cells.Western blot was used to further detect the expression of proliferation-related genes PCNA and CCND1.The results showed that overexpression of PITX2 significantly up-regulated the expression of protein levels of PCNA and CCND1;PITX2-siRNA significantly down-regulated the expression of protein levels of PCNA and CCND1.In summary,PITX2 activates the WNT/β-catenin signaling pathway by regulating the expression of CTNNB1,regulates the synthesis and secretion of E₂,P₄ in ovarian granulosa cells of dairy goat;promotes the proliferation of granulosa cells.The above research results provide a basis for further exploring the regulation mechanism of PITX2 on the follicular development and ovulation of dairy goats.