Zheng C Y, Zou X, Lin H J, Zhao B C, Zhang M L, Luo C H, Fu S X
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China.
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China.
Theriogenology. 2018 Apr 1;110:116-121. doi: 10.1016/j.theriogenology.2017.12.050. Epub 2018 Jan 9.
Retention of fetal membranes (RFM) of cows is an important reproductive disturbance, and is related to miRNAs. Vascular endothelial growth factor (VEGF)A, regulated by miRNA-185, can activate arachidonic acid (ARA) release via the VEGFA signaling pathway, which influences RFM. The aim of this study was to explore the pathogenic mechanism of RFM by investigating the regulatory relationship between miRNA-185 and the VEGFA signaling pathway. Serum samples of healthy Holstein dairy cows (n = 20) and RFM cows (n = 12), with a similar age, parity, weight, and milk yield, were collected to detect VEGFA and ARA concentrations at 6, 12, and 24 h after calving. Caruncle tissues were collected from healthy (n = 6) and RFM cows (n = 6) at 12 h after calving. Quantitative polymerase chain reaction (qPCR) and western blotting (WB) were performed to detect the mRNA and proteins levels, respectively, of genes involved in the VEGFA signaling pathway. Uterine caruncle epithelial (UCE) cells were cultured by the explant culture method, further purified, and subsequently treated with miRNA-185 mimics, miRNA-185 mimics + MEK inhibitor, or left untreated as a control for detection of the mRNA and protein levels of genes involved in the VEGFA signaling pathway. The cellular supernatant was collected for measurement of ARA levels at 12, 24 and 48 h after treatment. Serum levels of VEGFA and ARA from RFM cows were abnormally increased at 12 h after calving, as compared to those in healthy dairy cows. Expression levels of most of the investigated genes (VEGFA, PLC, PRK, RAF, MEK, MAPK, and PLA) were down-regulated in the caruncle tissue of RFM cows. However, P-p44/42 MAPK was up-regulated in the caruncle tissues of cows with RFM (p < .01). In UCE cells treated with the miRNA-185 mimics, expression of VEGFA, PLC, RAF, MEK, MAPK and PLA was significantly down-regulated, while that of P-p44/42 MAPK was significantly up-regulated. Expression of genes involved in the VEGFA signaling pathway was similar to that in the in vivo assay. In UCE cells treated with the miRNA-185 mimics + MEK inhibitors, expression of VEGFA, PLC, RAF, MEK, MAPK and P-p44/42 MAPK was significantly down-regulated, while that of PLA was significantly up-regulated. Meanwhile, the release of ARA was increased (p < .01). These results demonstrate that miRNA-185 can regulate the VEGFA signaling pathway, especially via abnormal expression of P-p44/42 MAPK, which influences the release of the fetal placenta after calving.
奶牛胎膜滞留（RFM）是一种重要的繁殖障碍，与微小RNA（miRNA）有关。受miRNA - 185调控的血管内皮生长因子（VEGF）A可通过VEGFA信号通路激活花生四烯酸（ARA）释放，这会影响胎膜滞留。本研究的目的是通过研究miRNA - 185与VEGFA信号通路之间的调控关系，探讨胎膜滞留的致病机制。收集年龄、胎次、体重和产奶量相似的健康荷斯坦奶牛（n = 20）和胎膜滞留奶牛（n = 12）的血清样本，以检测产犊后6、12和24小时的VEGFA和ARA浓度。在产犊后12小时，从健康奶牛（n = 6）和胎膜滞留奶牛（n = 6）收集肉阜组织。分别进行定量聚合酶链反应（qPCR）和蛋白质免疫印迹法（WB），以检测VEGFA信号通路相关基因的mRNA和蛋白质水平。采用组织块培养法培养子宫肉阜上皮（UCE）细胞，进一步纯化，随后用miRNA - 185模拟物、miRNA - 185模拟物 + MEK抑制剂处理，或不处理作为对照，以检测VEGFA信号通路相关基因的mRNA和蛋白质水平。处理后12、24和48小时收集细胞上清液，用于测定ARA水平。与健康奶牛相比，胎膜滞留奶牛产犊后12小时血清VEGFA和ARA水平异常升高。在胎膜滞留奶牛的肉阜组织中，大多数研究基因（VEGFA、PLC、PRK、RAF、MEK、MAPK和PLA）的表达水平下调。然而，在胎膜滞留奶牛的肉阜组织中，P - p44/42 MAPK上调（p < 0.01）。在用miRNA - 185模拟物处理的UCE细胞中，VEGFA、PLC、RAF、MEK、MAPK和PLA的表达显著下调，而P - p44/42 MAPK的表达显著上调。VEGFA信号通路相关基因的表达与体内试验相似。在用miRNA - 185模拟物 + MEK抑制剂处理的UCE细胞中，VEGFA、PLC、RAF、MEK、MAPK和P - p44/42 MAPK的表达显著下调，而PLA的表达显著上调。同时，ARA的释放增加（p < 0.01）。这些结果表明，miRNA - 185可以调节VEGFA信号通路，尤其是通过P - p44/42 MAPK的异常表达，这会影响产犊后胎儿胎盘的释放。
