猪miR-192靶向调控Wnt7a基因表达的研究

doi:10.16431/j.cnki.1671-7236.2024.06.005

摘要/Abstract

摘要： 【目的】鉴定猪miR-192与Wnt家族成员7A(Wnt7a)基因之间的靶向关系,为进一步构建miR-192介导的胚胎通讯提供依据。【方法】使用miRNA pull-down鉴定miR-192和Wnt7a基因间的互作关系;利用生物信息学软件RNAhybrid 2.2预测miR-192和Wnt7a基因3'-UTR的结合位点,构建含有miR-192结合位点的Wnt7a基因3'-UTR野生型和突变型基因片段,克隆至双荧光素酶报告基因质粒,通过NotⅠ、XhoⅠ酶切和测序进行鉴定;鉴定成功的质粒分别与miR-192 mimics和mimics NC共转染猪子宫内膜上皮细胞,检测双荧光素酶活性;将miR-192 mimics、mimics NC、miR-192 inhibitor、inhibitor NC分别与构建的Wnt7a基因3'-UTR野生型和突变型双荧光素酶报告基因质粒共转染至猪子宫内膜上皮细胞,通过实时荧光定量PCR和Western blotting检测Wnt7a基因mRNA和蛋白的表达水平。【结果】pull-down结果显示,miR-192 mimics组与其阴性对照组相比,Wnt7a基因相对表达量极显著升高(P＜0.01),miR-192 input组相对于其阴性对照组极显著降低(P＜0.01);RNAhybrid 2.2软件预测到miR-192种子区与Wnt7a基因3'-UTR存在结合位点(UGACCUA);转染Wnt7a野生型质粒的miR-192 mimics和miR-192 mimics NC组相比双荧光素酶活性显著降低(P＜0.05),转染Wnt7a突变型质粒的miR-192 mimics和miR-192 mimics NC组之间双荧光素酶活性无显著差异(P＞0.05)。实时荧光定量PCR结果表明,miR-192 mimics组Wnt7a基因mRNA相对表达量显著低于其阴性对照组(P＜0.05),miR-192 inhibitor组显著高于其阴性对照组(P＜0.05)。Western blotting结果表明,过表达miR-192极显著抑制了Wnt7a蛋白的表达(P＜0.01),抑制miR-192后Wnt7a蛋白的表达量极显著上升(P＜0.01)。【结论】miR-192能够与Wnt7a基因3'-UTR发生靶向作用并抑制其表达。研究结果可为今后深入研究miR-192在妊娠着床过程中的调控机制提供理论依据。

关键词: 猪; miR-192; Wnt7a基因; 胚胎附植

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

中图分类号:

S828

符彬彬, 王东升, 何凡, 李清春, 祁梦凡, 张化鹏, 黄涛. 猪miR-192靶向调控Wnt7a基因表达的研究[J]. 中国畜牧兽医, 2024, 51(6): 2300-2307.

FU Binbin, WANG Dongsheng, HE Fan, LI Qingchun, QI Mengfan, ZHANG Huapeng, HUANG Tao. Study on the Targeted Regulation of Wnt7a Gene Expression by miR-192 in Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(6): 2300-2307.

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