Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (15): 3083-3092.doi: 10.3864/j.issn.0578-1752.2024.15.013

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

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

LIU BingQi(), LUO ChunHai, YAO WeiJia, WANG Wei, LIU JiaJin, LI DanYang, FU ShiXin()   

  1. College of Animal Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163316, Heilongjiang
  • Received:2024-02-05 Accepted:2024-05-07 Online:2024-08-05 Published:2024-08-05
  • Contact: FU ShiXin

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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

Table 1

Primers of miRNA-424-5p and reference gene"

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

Table 2

Primer sequences"

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

Fig. 1

Expression levels of miRNA-424-5p and VEGFA-related genes N: Healthy group cows; R:Cows with RFM. A. The mRNA expression of miRNA-424-5p in maternal placenta was detected; B. The mRNA expression changes of VEGFA, MMP-2, MMP-9 and COL-IV in maternal placenta were detected; C. The expression changes of VEGFA, MMP-2, MMP-9 and COL-IV proteins in maternal placenta were detected"

Fig. 2

Results of miRNA-424-5p target gene prediction and validation of its relationship with VEGFA targeting A. miRNA-424-5p predicted the number of target genes; B. Results of double luciferase experiment"

Fig. 3

Effect of overexpression and silencing of miRNA424-5p on VEGFA expression A. VEGFA mRNA expression was detected; B. VEGFA protein expression was detected"

Fig. 4

The effect of miRNA-424-5p on VEGFA expression in endometrial epithelial cells was detected by immunofluorescence assay"

Fig. 5

Effects of overexpression and silencing of miRNA-424-5p on the expression of MMP-2, MMP-9 and COL-IV A. mRNA expression changes of MMP-2, MMP-9 and COL-IV; B. Protein expression changes of MMP-2, MMP-9 and COL-IV"

[1]
HOOSHMANDABBASI R, ZERBE H, BAUERSACHS S, DE SOUSA N M, BOOS A, KLISCH K. Pregnancy-associated glycoproteins in cows with retained fetal membranes. Theriogenology, 2018, 105: 158-163.

doi: S0093-691X(17)30462-4 pmid: 28982025
[2]
于程, 汤兆学. 奶牛胎衣不下的病因、临床症状和防治措施. 现代畜牧科技, 2018(3): 63.
YU C, TANG Z X. Etiology, clinical symptoms and prevention measures of retained placenta in dairy cows. Modern Animal Husbandry Science & Technology, 2018(3): 63. (in Chinese)
[3]
GOHARY K, LEBLANC S J. Cost of retained fetal membranes for dairy herds in the United States. Journal of the American Veterinary Medical Association, 2018, 252(12): 1485-1489.

doi: 10.2460/javma.252.12.1485 pmid: 29889640
[4]
POHL A, BURFEIND O, HEUWIESER W. The associations between postpartum serum haptoglobin concentration and metabolic status, calving difficulties, retained fetal membranes, and metritis. Journal of Dairy Science, 2015, 98(7): 4544-4551.

doi: 10.3168/jds.2014-9181 pmid: 25912860
[5]
KUMARI S, PRASAD S, KUMARESAN A, MANIMARAN A, PATBANDHA T K, PATHAK R, BORO P, MOHANTY T K, RAVI S K. Risk factors and impact of retained fetal membranes on performance of dairy bovines reared under subtropical conditions. Tropical Animal Health and Production, 2015, 47(2): 285-290.

doi: 10.1007/s11250-014-0717-z pmid: 25377506
[6]
BARTEL D P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell, 2004, 116(2): 281-297.

doi: 10.1016/s0092-8674(04)00045-5 pmid: 14744438
[7]
QU Y, FADDEN A N, TRABER M G, BOBE G. Potential risk indicators of retained placenta and other diseases in multiparous cows. Journal of Dairy Science, 2014, 97(7): 4151-4165.

doi: 10.3168/jds.2013-7154 pmid: 24792789
[8]
DERVISHI E, ZHANG G S, HAILEMARIAM D, DUNN S M, AMETAJ B N. Occurrence of retained placenta is preceded by an inflammatory state and alterations of energy metabolism in transition dairy cows. Journal of Animal Science and Biotechnology, 2016, 7(1): 26.
[9]
RAPACZ-LEONARD A, DĄBROWSKA M, JANOWSKI T. Major histocompatibility complex I mediates immunological tolerance of the trophoblast during pregnancy and may mediate rejection during parturition. Mediators of Inflammation, 2014, 2014: 579279.
[10]
DILLY M, HAMBRUCH N, SHENAVAI S, SCHULER G, FROEHLICH R, HAEGER J D, OZALP G R, PFARRER C. Expression of matrix metalloproteinase (MMP)-2, MMP-14 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 during bovine placentation and at term with or without placental retention. Theriogenology, 2011, 75(6): 1104-1114.

doi: 10.1016/j.theriogenology.2010.11.019 pmid: 21247626
[11]
HANSEN P J. PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Maternal immunological adjustments to pregnancy and parturition in ruminants and possible implications for postpartum uterine health: Is there a prepartum-postpartum nexus? 1. Journal of Animal Science, 2013, 91(4): 1639-1649.

doi: 10.2527/jas.2012-5934 pmid: 23307838
[12]
ATTUPURAM N M, KUMARESAN A, NARAYANAN K, KUMAR H. Cellular and molecular mechanisms involved in placental separation in the bovine: A review. Molecular Reproduction and Development, 2016, 83(4): 287-297.

doi: 10.1002/mrd.22635 pmid: 26970238
[13]
GROSS T S, WILLIAMS W F. Bovine placental prostaglandin synthesis: principal cell synthesis as modulated by the binucleate cell. Biology of Reproduction, 1988, 38(5): 1027-1034.

pmid: 3165677
[14]
WANG Y H, WU N, PANG B, TONG D D, SUN D L, SUN H M, ZHANG C Y, SUN W J, MENG X N, BAI J, CHEN F, GENG J S, FU S B, JIN Y. TRIB1 promotes colorectal cancer cell migration and invasion through activation MMP-2 via FAK/Src and ERK pathways. Oncotarget, 2017, 8(29): 47931-47942.

doi: 10.18632/oncotarget.18201 pmid: 28624785
[15]
LEŚNIAK-WALENTYN A, HRABIA A. Expression and localization of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct during maturation. Cell and Tissue Research, 2016, 364(1): 185-197.
[16]
邢菲菲, 罗胜缤, 温小庆, 曹一鸣, 李美娟, 罗春海, 付世新. NEFA经MMPs途径对奶牛子宫内膜上皮细胞Ⅳ型胶原蛋白表达的影响. 中国兽医学报, 2022, 42(11): 2250-2255.
XING F F, LUO S B, WEN X Q, CAO Y M, LI M J, LUO C H, FU S X. Effects of NEFA on expression of type Ⅳ collagen in endometrial epithelial cells of dairy cows via MMPs pathway. Chinese Journal of Veterinary Science, 2022, 42(11): 2250-2255. (in Chinese)
[17]
DUMITRU C S, RAICA M. Vascular endothelial growth factor family and head and neck squamous cell carcinoma. Anticancer Research, 2023, 43(10): 4315-4326.
[18]
陈艳, 蔺婷, 向福兰, 周芳亮, 何迎春. VEGFA对肿瘤发生发展的作用及其机制的研究进展. 现代医学, 2023, 51(9): 1345-1352.
CHEN Y, LIN T, XIANG F L, ZHOU F L, HE Y C. Research progress on the role of VEGFA in tumor occurrence and development and its mechanism. Modern Medical Journal, 2023, 51(9): 1345-1352. (in Chinese)
[19]
CONNOLLY D T, HEUVELMAN D M, NELSON R, OLANDER J V, EPPLEY B L, DELFINO J J, SIEGEL N R, LEIMGRUBER R M, FEDER J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. Journal of Clinical Investigation, 1989, 84(5): 1470-1478.

doi: 10.1172/JCI114322 pmid: 2478587
[20]
ROSKOSKI R. ERK1/ 2 MAP kinases: Structure, function, and regulation. Pharmacological Research, 2012, 66(2): 105-143.
[21]
KAMADA H, MATSUI Y, SAKURAI Y, TANIGAWA T, ITOH M, KAWAMOTO S, KAI K, SASAKI T, TAKAHASHI K, HAYASHI M, TAKAYAMA Y, NAKAMURA M, KADOKAWA H, UEDA Y, SUTOH M, MURAI M. Twelve oxo-eicosatetraenoic acid induces fetal membrane release after delivery in cows. Placenta, 2012, 33(2): 106-113.

doi: 10.1016/j.placenta.2011.11.001 pmid: 22118869
[22]
郑程远. 胎衣不下奶牛母体胎盘组织差异表达microRNA的筛选及验证[D]. 大庆: 黑龙江八一农垦大学, 2015.
ZHENG C Y. Filter and verify differential expresseion of microRNAs in the maternal placenta of cattle with retained foetal membranes[D]. Daqing: Heilongjiang Bayi Agricultural University, 2015. (in Chinese)
[23]
邹晓. MiRNA-185调控VEGFA/STIM1表达与奶牛胎衣不下的关系[D]. 大庆: 黑龙江八一农垦大学, 2016.
ZOU X. The relationship between miRNA-185 regulates VEGFA/ STIM1 genes and the cattle with retained foetal membranes[D]. Daqing: Heilongjiang Bayi Agricultural University, 2016. (in Chinese)
[24]
AMIN Y A, HUSSEIN H A. Latest update on predictive indicators, risk factors and ‘Omic’ technologies research of retained placenta in dairy cattle - A review. Reprod Domest Anim, 2022, 57(7): 687-700.
[25]
JAGLAN K, DHAKA S S, MAGOTRA A, PATIL C S, GHANGHAS A. Exploring microRNA biogenesis, applications and bioinformatics analysis in livestock: a comprehensive review. Reproduction in Domestic Animals, 2024, 59(1): e14529.
[26]
陈玉梅, 张聪聪, 胡丽蓉, 房浩, 窦金焕, 郭刚, 王炎, 刘巧香, 王雅春, 徐青. 热应激对奶牛GNAS启动子区DNA甲基化水平的影响. 中国农业科学, 2023, 56(12): 2395-2406.

doi: 10.3864/j.issn.0578-1752.2023.12.013
CHEN Y M, ZHANG C C, HU L R, FANG H, DOU J H, GUO G, WANG Y, LIU Q X, WANG Y C, XU Q. Effect of heat stress on DNA methylation of GNAS promoter region in dairy cows. Scientia Agricultura Sinica, 2023, 56(12): 2395-2406. (in Chinese)
[27]
LIU M Z, CHEN J H, ZHANG C L, LIU S H, CHAO X H, YANG H, MUHAMMAD A, ZHOU B, AO W P, SCHINCKEL A P. Deciphering estrus expression in gilts: The role of alternative polyadenylation and LincRNAs in reproductive transcriptomics. Animals, 2024, 14(5): 791.
[28]
PANG X L, LI J, WANG J, YAN S S, YANG J. MiR-142-3p regulates ILC1s by targeting HMGB1 via the NF-κB pathway in a mouse model of early pregnancy loss. Current Medical Science, 2024, 44(1): 195-211.
[29]
郑程远. miRNA-185调控VEGFA信号通路影响奶牛胎衣不下的分子机制[D]. 大庆: 黑龙江八一农垦大学, 2018.
ZHENG C Y. miRNA-185 regulates the VEGFA signaling pathway in dairy cows with retained fetal membranes[D]. Daqing: Heilongjiang Bayi Agricultural University, 2018. (in Chinese)
[30]
GRZESIAK M, KAMINSKA K, KNAPCZYK-STWORA K, HRABIA A. The expression and localization of selected matrix metalloproteinases (MMP-2, -7 and-9) and their tissue inhibitors (TIMP-2 and -3) in follicular cysts of sows. Theriogenology, 2022, 185: 109-120.
[31]
TANAKA N, SAKAMOTO T. MT1-MMP as a key regulator of metastasis. Cells, 2023, 12(17): 2187.
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