摘要
【目的】探讨孕酮对奶牛子宫内膜上皮细胞容受性标志基因表达的影响,为研究孕酮与子宫内膜容受性的关系及其调控机制提供理论依据。【方法】用0、10、100μg/L孕酮处理奶牛子宫内膜上皮细胞,通过显微镜观察细胞形态,利用CCK8试验检测细胞增殖水平,使用实时荧光定量PCR检测磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)、血管内皮生长因子(VEGF)和同源盒结构基因10(HOXa10)的mRNA表达水平,筛选孕酮最佳作用浓度。用孕酮、转录激活因子3(STAT3)抑制剂(1.5μmol/L)、孕酮+STAT3抑制剂分别处理奶牛子宫内膜上皮细胞,通过实时荧光定量PCR和Western blotting分别检测VEGF和HOXa10的mRNA和蛋白表达水平。使用Lipofectamine 3000将VEGF的靶向miRNA(miR-497 mimic、miR-497 inhibitor)、HOXa10的靶向miRNA(miR-27a-3p mimic、miR-27a-3p inhibitor)以及阴性对照(mimic-NC、inhibitor-NC)分别转染奶牛子宫内膜上皮细胞,采用实时荧光定量PCR检测细胞中miR-497、miR-27a-3p以及VEGF和HOXa10的表达情况。【结果】与0μg/L组相比,10和100μg/L孕酮处理组细胞增殖水平、PI 3 K和Akt基因的mRNA表达水平没有显著差异(P>0.05),VEGF和HOXa 10基因mRNA表达水平显著升高(P<0.05),后续选用10μg/L孕酮进行试验。与10μg/L孕酮处理组相比,STAT3抑制剂处理组和孕酮+STAT3抑制剂联合处理组细胞中VEGF和HOXa10的mRNA和蛋白表达水平均显著下降(P<0.05),但与对照组无显著差异(P>0.05)。与mimic-NC组相比,转染miR-497 mimic、miR-27a-3p mimic后细胞中VEGF、HOXa10的mRNA及蛋白表达水平均显著下降(P<0.05);与inhibitor-NC组相比,转染miR-497 inhibitor、miR-27a-3p inhibitor后细胞中VEGF、HOXa10的mRNA及蛋白表达水平均显著升高(P<0.05)。【结论】孕酮通过激活STAT3信号通路调节奶牛子宫内膜上皮细胞中VEGF和HOXa 10基因的表达,miR-497负调控VEGF基因的表达,miR-27a-3p负调控HOXa 10基因的表达。
【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 100μg/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(1.5μmol/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 0μg/L group,there were no significant differences in cell proliferation level,mRNA expression of PI 3 K and Akt genes in 10 and 100μg/L progesterone treatment groups(P>0.05),while VEGF and HOXa 10 genes mRNA expression were significantly increased(P<0.05).Subsequently,10μg/L progesterone was selected for the test.Compared with 10μg/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 HOXa 10 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 HOXa 10 gene.
作者
唐颖
李琦
王红战
李博
陈艳茹
郑鹏
TANG Ying;LI Qi;WANG Hongzhan;LI Bo;CHEN Yanru;ZHENG Peng(College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2024年第11期4711-4721,共11页
China Animal Husbandry & Veterinary Medicine
基金
“十四五”国家重点研发计划课题(2023YFD1300602)
黑龙江省现代农业奶牛产业技术协同创新推广体系(230000237613110000244)。
关键词
孕酮
奶牛子宫内膜上皮细胞
容受性基因
progesterone
bovine endometrial epithelial cells
receptivity gene
