miRNA-424-5p靶向VEGFA对胎衣不下奶牛胎盘胶原蛋白降解的影响

Mechanism of miRNA-424-5p Regulate the Decomposition of Collagen on Retained Fetal Membranes of Dairy Cows Through VEGFA Pathway

【目的】通过探究miRNA-424-5p对奶牛母体胎盘组织胶原蛋白降解的影响,进而明确miRNA-424-5p对奶牛胎衣不下(retained fetal membranes,RFM)发生的调控作用及机制。【方法】检测胎衣正常排出与胎衣不下奶牛母体胎盘组织miRNA-424-5p表达水平,采用qRT-PCR和Western blot方法进行胎衣正常排出与胎衣不下奶牛的母体胎盘组织VEGFA、MMP-2、MMP-9、COL-IV的表达水平检测。应用生物信息学分析对miRNA-424-5p进行靶基因预测,并通过双荧光素酶试验验证miRNA-424-5p与VEGFA的靶向关系。在奶牛子宫内膜上皮细胞中转染miRNA-424-5p mimics和miRNA-424-5p inhibitor进行过表达和沉默miRNA-424-5p。采用免疫荧光技术观察VEGFA在奶牛子宫内膜上皮细胞的表达变化,qRT-PCR和Western blot检测VEGFA、MMP-2、MMP-9、COL-IV的mRNA与蛋白表达水平的变化。【结果】与健康组相比,RFM组母体胎盘组织中的miRNA-424-5p mRNA表达水平显著升高(P<0.01),VEGFA、MMP-2、MMP-9 mRNA与蛋白表达水平极显著降低(P<0.01),COL-IV mRNA与蛋白表达水平极显著升高(P<0.01)。miRNA-424-5p的潜在靶基因共有152个,并经双荧光素酶试验结果证实VEGFA是miRNA-424-5p的靶基因。过表达和沉默miRNA-424-5p后,免疫荧光结果显示,与对照组相比miRNA-424-5p mimics组VEGFA的表达较低,而miRNA-424-5p inhibitor组VEGFA表达较高;qRT-PCR和Western blot结果显示,过表达miRNA-424-5p,靶基因VEGFA mRNA与蛋白表达水平极显著降低,基质金属蛋白酶MMP-2、MMP-9 mRNA与蛋白表达水平极显著降低(P<0.01),胶原蛋白COL-IV mRNA与蛋白表达水平极显著升高(P<0.01),而沉默miRNA-424-5p时VEGFA、MMP-2、MMP-9 mRNA与蛋白表达显著升高(P<0.01),COL-IV mRNA与蛋白表达水平极显著降低。【结论】miRNA-424-5p可靶向VEGFA调控MMPs表达、胶原蛋白的分解从而引起细胞外胶原降解障碍,是引起胎衣不下发生的重要因素。

【Objective】To investigate the effect of miRNA-424-5p on collagen degradation in placental tissues of dairy cows,and then to clarify the regulatory role and mechanism of miRNA-424-5p on the occurrence of retained fetal membranes(RFM)in dairy cows.【Method】The expression level of miRNA-424-5p in maternal placenta tissues of normal cows and cows with RFM were firstly detected,and the expression levels of VEGFA,MMP-2,MMP-9 and COL-IV in maternal placenta tissues of normal cows and cows with RFM were detected by qRT-PCR and Western blot.Prediction of target genes for miRNA-424-5p was performed using bioinformatics analysis,the targeting relationship of miRNA-424-5p with VEGFA was verified using the dual luciferase assay.miRNA-424-5p mimics and miRNA-424-5p inhibitor were transfected in dairy endometrial epithelial cells for overexpression and silencing of miRNA-424-5p.Immunofluorescence was used to observe the changes of VEGFA expression in dairy endometrial epithelial cells.The expression of VEGFA,MMP-2,MMP-9 and COL-IV were detected by qRT-PCR and Western blot.【Result】Compared with the healthy group,miRNA-424-5p mRNA expression level in maternal placental tissues of RFM group was significantly increased(P<0.01),the expression of VEGFA,MMP-2,MMP-9 mRNA and protein was highly significant decreased(P<0.01).COL-IV mRNA and protein was highly significant increased(P<0.01).There were 152 potential target genes of miRNA-424-5p,and VEGFA was confirmed to be the target gene of miRNA-424-5p by the results of dual luciferase assay.After overexpression and silencing of miRNA-424-5p,the immunofluorescence results showed that the expression of VEGFA was lower in the miRNA424-5p mimics group compared with the control group,while the expression of VEGFA was higher in the miRNA424-5p inhibitor group.The qRT-PCR and Western blot results showed that overexpression of miRNA-424-5p,the expression of target gene VEGFA mRNA and protein were significantly decreased,and the expression of MMP-2 and MMP-9 mRNA and protein were highly significantly down-regulated(P<0.01),and COL-IV mRNA and protein expression levels were highly significantly increased(P<0.01).【Conclusion】miRNA-424-5p can through VEGFA pathway to regulate the degradation of MMPs and collagen,and can cause the obstacle of extracellular collagen degradation,miRNA-424-5p is an important factor of causing the occurrence of retained fetal membranes.