Study on Regulation of Receptivity Gene Expression in Bovine Endometrial Epithelial Cells by Progesterone
[Objective]This study was aimed to explore the effect of progesterone on the expression of receptivity marker genes in bovine endometrial epithelial cells,and provide theoretical basis for studying the relationship between progesterone and endometrial receptivity and its regulatory mechanism.[Method]Bovine endometrial epithelial cells were treated with 0,10 and 100 µg/L progesterone,and the cell morphology was observed by microscope.The cell proliferation level was detected by CCK8 assay,and the mRNA expression of phosphatidylinositol 3 kinase(PI3K),protein kinase B(Akt),vascular endothelial growth factor(VEGF)and homeobox structure gene 10(HOXa10)were detected by Real-time quantitative PCR,and the optimal concentration of progesterone was screened.Bovine endometrial epithelial cells were treated with progesterone,transcription activator 3(STAT3)inhibitor(1.5 μmol/L),and progesterone+STAT3 inhibitor,respectively.The mRNA and protein expression of VEGF and HOXa10 were detected by Real-time quantitative PCR and Western blotting,respectively.With Lipofectamine 3000,VEGF targeting miRNA(miR-497 mimic and miR-497 inhibitor)and HOXa10 targeting miRNA(miR-27a-3p mimic and miR-27a-3p inhibitor)and negative control(mimic-NC and inhibitor-NC)were transfected into endometrial epithelial cells,respectively.Real-time quantitative PCR was used to detect the expression of miR-497,miR-27a-3p,VEGF and HOXa10.[Result]Compared with 0 μg/L group,there were no significant differences in cell proliferation level,mRNA expression of PI3K and Akt genes in 10 and 100 μg/L progesterone treatment groups(P＞0.05),while VEGF and HOXa10 genes mRNA expression were significantly increased(P＜0.05).Subsequently,10 μg/L progesterone was selected for the test.Compared with 10 μg/L progesterone treatment group,the mRNA and protein expression of VEGF and HOXa10 in STAT3 inhibitor treatment group and progesterone+STAT3 inhibitor combined treatment group were significantly decreased(P＜0.05),but there was no significant difference compared with control group(P＞0.05).Compared with mimic-NC group,mRNA and protein expression of VEGF and HOXa10 in cells transfected with miR-497 mimic and miR-27a-3p mimic were significantly decreased(P＜0.05).Compared with inhibitor-NC group,mRNA and protein expression of VEGF and HOXa10 in cells transfected with miR-497 inhibitor and miR-27a-3p inhibitor were significantly increased(P＜0.05).[Conclusion]Progesterone regulated the expression of VEGF and HOXa10 genes in bovine endometrial epithelial cells by activating STAT3 signaling pathway,miR-497 negatively regulated the expression of VEGF gene,and miR-27a-3p negatively regulated the expression of HOXa10 gene.
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