胎衣不下奶牛miRNA-185靶向调控血管内皮生长因子A表达的研究

doi:10.16431/j.cnki.1671-7236.2024.03.003

摘要/Abstract

摘要： 【目的】通过体内和体外试验检测miRNA-185及血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)在胎衣正常排出和胎衣不下(RFM)奶牛胎盘组织中的差异表达情况及VEGFA在奶牛母体胎盘组织中表达分布,验证及明确miRNA-185和VEGFA与奶牛胎衣不下发生密切相关并存在靶向调节关系,为深入研究miRNA-185在奶牛胎衣不下发生过程中的作用提供理论依据和试验基础。【方法】采用实时荧光定量PCR法检测胎衣正常排出及胎衣不下奶牛母体胎盘组织中miRNA-185及VEGFA基因表达情况,采用荧光原位杂交技术(FISH)检测VEGFA在母体胎盘组织的表达分布及mRNA的差异表达情况;采用双荧光素酶明确miRNA-185与VEGFA的靶向调节关系,采用实时荧光定量PCR及Western blotting检测体外转染miRNA185 mimics、miRNA-185 inhibitor及miRNA-185 NC后VEGFA mRNA及蛋白的差异表达情况。【结果】实时荧光定量PCR结果显示,与胎衣正常排出奶牛相比,胎衣不下奶牛体内miRNA-185及VEGFA的表达水平均极显著下调(P＜0.01);荧光原位杂交结果显示,VEGFA mRNA主要在奶牛子宫内膜上皮细胞中表达。VEGFA是miRNA-185的靶向调节蛋白。与阴性对照组相比,转染miRNA-185 mimics后VEGFA的mRNA及蛋白表达水平均极显著下调(P＜0.01),转染miRNA-185 inhibitor后VEGFA的mRNA及蛋白表达水平均极显著上调(P＜0.01)。【结论】胎衣不下奶牛母体胎盘组织内miRNA-185及VEGFA表达显著降低,VRGFA主要在子宫内膜上皮细胞中表达,miRNA-185可通过靶向调控VEGFA的表达参与奶牛胎衣不下的发生发展。

关键词: 奶牛; 胎衣不下; miRNA-185; 血管内皮生长因子A; 母体胎盘

Abstract: 【Objective】 This experiment was aimed to detect the differential expression of miRNA-185 and vascular endothelial growth factor A (VEGFA) in the placental tissue of dairy cows in the normal expulsion group and retained fetal membranes (RFM) group through in vivo and in vitro experiments,as well as the expression distribution of VEGFA in the placental tissue of dairy cows.It verified and clarified the close correlation and targeted regulatory relationship between miRNA-185 and VEGFA and retained fetal membranes in dairy cows and provided theoretical and experimental basis for the in-depth study of the role of miRNA-185 in the occurrence of retained fetal membranes in dairy cows.【Method】 Real-time fluorescence quantitative PCR method was used to detect the gene expression of miRNA-185 and VEGFA in the placental tissue of dairy cows with normal and retained fetal membranes.Fluorescence in situ hybridization (FISH) was used to detect the specific and mRNA differential expression of VEGFA in the placental tissue of dairy cows.Dual luciferase reporter genes was used to clarify the targeted regulatory relationship between miRNA-185 and VEGFA,Real-time fluorescence quantitative PCR and Western blotting methods were used to detect the mRNA and protein differential expression of VEGFA after in vitro transfection of miRNA185 mimics,miRNA-185 inhibitor and miRNA-185 NC.【Result】 Real-time fluorescence quantitative PCR results showed that compared with dairy cows with normal removal of placenta,the expression levels of miRNA-185 and VEGFA in dairy cows with retained fetal membranes were extreme downregulation (P＜0.01).FISH result showed that VEGFA mRNA was mainly expressed in dairy cow endometrial epithelial cells.VEGFA was a targeted regulatory protein of miRNA-185.Compared with the negative control group,the mRNA and protein expression levels of VEGFA were extreme downregulation after transfection with miRNA-185 mimics (P＜0.01),while the mRNA and protein expression levels of VEGFA were extremely upregulated after transfection with miRNA-185 inhibitor (P＜0.01).【Conclusion】 The expression of miRNA-185 and VEGFA in the placental tissue of dairy cows with retained fetal membranes significantly decreased.VRGFA was mainly expressed in endometrial epithelial cells,and miRNA-185 could participate in the occurrence and development of retained fetal membranes in dairy cows by targeting and regulating the expression of VEGFA.

Key words: dairy cows; retained fetal membranes; miRNA-185; vascular endothelial growth factor A; maternal placenta

中图分类号:

S858.23

罗春海, 郑程远, 张梦龙, 姚伟佳, 刘佳金, 刘炳琦, 王薇, 付世新. 胎衣不下奶牛miRNA-185靶向调控血管内皮生长因子A表达的研究[J]. 中国畜牧兽医, 2024, 51(3): 916-925.

LUO Chunhai, ZHENG Chengyuan, ZHANG Menglong, YAO Weijia, LIU Jiajin, LIU Bingqi, WANG Wei, FU Shixin. Regulation of Vascular Endothelial Growth Factor A Expression by miRNA-185 in Dairy Cows with Retained Fetal Membranes[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 916-925.

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