miRNA-424-5p靶向VEGFA对胎衣不下奶牛胎盘胶原蛋白降解的影响

doi:10.3864/j.issn.0578-1752.2024.15.013

摘要/Abstract

摘要：

【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。

关键词: 奶牛, 胎衣不下, miRNA-424-5p, 血管内皮生长因子A, 胶原蛋白降解

Abstract:

【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.

Key words: cow, retained fetal membranes, miRNA-424-5p, vascular endothelial growth factor A, collagen degradation

刘炳琦, 罗春海, 姚伟佳, 王薇, 刘佳金, 李丹阳, 付世新. miRNA-424-5p靶向VEGFA对胎衣不下奶牛胎盘胶原蛋白降解的影响[J]. 中国农业科学, 2024, 57(15): 3083-3092.

LIU BingQi, LUO ChunHai, YAO WeiJia, WANG Wei, LIU JiaJin, LI DanYang, FU ShiXin. Mechanism of miRNA-424-5p Regulate the Decomposition of Collagen on Retained Fetal Membranes of Dairy Cows Through VEGFA Pathway[J]. Scientia Agricultura Sinica, 2024, 57(15): 3083-3092.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.15.013

https://www.chinaagrisci.com/CN/Y2024/V57/I15/3083

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引物名称Primer	引物序列 Sequence（5′ to 3′）
miRNA-424-5p	RT:GTCGTATCCAGTGCAGGGTCCGAGGTGCACTGGATACGACTCAAAACA
	F:TGCGGCAGCAGCAATTCATG
	R:CAGTGCAGGGTCCGAGGT
U6	RT:CGCTTCACGAATTTGCGTGTCAT
	F:GCTTCGGCAGCACATATACTAAAAT
	R:CGCTTCACGAATTTGCGTGTCAT

基因 Gene	序列 Sequences（5′-3′）
VEGFA	F:CCCACGAAGTGGTGAAGTTCA
VEGFA	R:CCACCAGGGTCTCGATGG
MMP-2	F:GGACCCTGGAGCCCTGATGG
MMP-2	R:GAGTTCTTGGATGCCCTGGATGTC
MMP-9	F:GGTGCTGGCTTGCTGCTCTG
MMP-9	R:TTGGTGAGGTTGGTTCGTGGTTC
COL-IV	F:TCCAGGGTTTCCAGGCGACTC
COL-IV	R:GGTCCCGTGCCAATAACAGTTCC
β-actin	F:TCCTGCGGCATTCACGAAACTAC
β-actin	R:GTGTTGGCGTAGAGGTCCTTGC