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