China Animal Husbandry & Veterinary Medicine ›› 2024, Vol. 51 ›› Issue (6): 2300-2307.doi: 10.16431/j.cnki.1671-7236.2024.06.005

• Biotechnology • Previous Articles     Next Articles

Study on the Targeted Regulation of Wnt7a Gene Expression by miR-192 in Pigs

FU Binbin1, WANG Dongsheng3, HE Fan1, LI Qingchun1, QI Mengfan1, ZHANG Huapeng1, HUANG Tao1,2   

  1. 1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
    2. Xinjiang Tiankang Animal Science and Technology Co., Ltd., Xinjiang Pig Breeding Engineering and Technology Research Center, Changji 831100, China;
    3. Xinjiang Production and Construction Corps Eighth Division Shihezi General Farm Agricultural Development Service Center, Shihezi 832000, China
  • Received:2023-11-02 Online:2024-06-05 Published:2024-06-01

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Abstract: 【Objective】 The targeting relationship between procine miR-192 and Wnt family member 7A (Wnt7a) genes was identified to establish a basis for further construction of miR-192-mediated embryonic communication.【Method】 The miRNA pull-down was used to identify the interactions between miR-192 and Wnt7a gene.Bioinformatics software RNAhybrid 2.2 was utilized to predict the binding sites of miR-192 and Wnt7a gene 3'-UTR.Fragments of Wnt7a gene 3'-UTR containing miR-192 binding sites were cloned into the dual luciferase reporter gene plasmid and subjected to identification through NotⅠ and XhoⅠ digestion and sequencing.The identified plasmids were co-transfected with miR-192 mimics and mimics NC into porcine endometrial epithelial cells to detect dual-luciferase activity.miR-192 mimics,mimics NC,miR-192 inhibitor,inhibitor NC were separately co-transfected with constructed Wnt7a gene 3'-UTR wild-type and mutant dual-luciferase reporter gene plasmids into porcine endometrial epithelial cells.The mRNA and protein expression of Wnt7a gene were detected by Real-time quantitative PCR and Western blotting.【Result】 Pull-down results showed that the relative expression of Wnt7a gene was extremely significantly increased in miR-192 mimics group compared with its negative control group (P＜0.01),and extremely significantly decreased in the miR-192 input group compared with its negative control group (P＜0.01).The binding site (UGACCUA) between miR-192 seed region and Wnt7a gene 3'-UTR was predicted by RNAhybrid 2.2 software.The dual luciferase activity was significantly lower in miR-192 mimics group transfected with Wnt7a wild-type plasmid compared with miR-192 mimics NC group (P＜0.05),and there was no significant difference in dual luciferase activity between miR-192 mimics group transfected with Wnt7a mutant plasmid and miR-192 mimics NC group (P＞0.05).Real-time quantitative PCR results showed that the mRNA relative expression of Wnt7a gene in miR-192 mimics group was significantly decreased than that in its negative control group (P＜0.05),and miR-192 inhibitor group was significantly higher than that in its negative control group (P＜0.05).Western blotting results showed that the expression of Wnt7a protein was extremely significant inhibited after overexpression of miR-192 (P＜0.01),and the expression of Wnt7a protein was extremely significant increased after inhibiting miR-192 (P＜0.01).【Conclusion】miR-192 was able to target Wnt7a gene 3'-UTR and inhibit its expression.The results of this study could provide a theoretical basis for future in-depth research on the regulatory mechanism of miR-192 in the process of pregnancy implantation.

Key words: pig; miR-192; Wnt7a gene; embryo attachment

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