bta-miR-377靶向调控牛下丘脑CART基因表达的研究

doi:10.16431/j.cnki.1671-7236.2022.09.004

摘要/Abstract

摘要： 【目的】分析bta-miR-377与牛卵泡发育的关键调控因子可卡因-苯丙胺调节转录肽（cocaine and amphetamine regulated transcript peptide，CART）间的靶标关系，明确bta-miR-377对牛CART表达影响的作用机制。【方法】本研究通过生物信息学手段预测bta-miR-377与牛CART基因mRNA的结合位点，并对miR-377在不同物种间的序列保守性进行分析；利用双荧光素酶报告基因法验证bta-miR-377与CART基因的靶标关系；选择3头健康西门塔尔母牛，采集其下丘脑组织，分析bta-miR-377和CART基因在牛下丘脑中的内源表达情况；以PC12和293T细胞为模型进行细胞功能验证，利用实时荧光定量PCR和Western blotting检测分别转染bta-miR-377 mimics和NC mimics后的PC12细胞中CART基因mRNA和293T细胞中CART蛋白表达丰度变化。【结果】 bta-miR-377在CART 3'-UTR上存在靶向结合位点，并能极显著下调CART 3'-UTR野生型重组双荧光质粒的相对荧光活性（P<0.01），且结合位点序列在多个物种中高度保守；在牛与大鼠间，miR-377种子区序列保守性较低；bta-miR-377和CART在牛下丘脑中均有表达；过表达bta-miR-377 mimics能够极显著下调CART基因mRNA的表达（P<0.01），显著下调CART蛋白的表达（P<0.05），bta-miR-377与CART的表达存在负相关关系。【结论】 bta-miR-377和CART在牛下丘脑组织中均有表达，bta-miR-377能够通过与CART 3'-UTR特异性结合抑制PC12细胞中CART mRNA的表达。

关键词: 牛; 下丘脑; CART; bta-miR-377

Abstract: 【Objective】 This study was aimed to analyze the interaction between bta-miR-377 and cocaine and amphetamine regulated transcript peptide(CART), a key regulatory factor of bovine follicle development, and to clarify the mechanism of bta-miR-377 on the expression of CART.【Method】 In this study, the binding sites of bta-miR-377 and bovine CART gene mRNA were predicted by bioinformatics, and the sequence conservation of miR-377 in different species was analyzed.The target relationship between bta-miR-377 and CART gene was verified by double luciferase reporter gene method.Three healthy Simmental cows were selected to collect their hypothalamic tissues and analyze the endogenous expression of bta-miR-377 and CART gene in the hypothalamus.PC12 and 293T cells were used as models for cell function verification.The abundance of CART gene mRNA in PC12 cells and CART protein in 293T cells transfected with bta-miR-377 mimics and NC mimics were detected by Real-time quantitative PCR and Western blotting, respectively.【Result】 bta-miR-377 had binding site to CART 3'-UTR and extremely significantly down-regulated the relative fluorescence activity of wild-type recombinant double fluorescent plasmid (P<0.01).The binding site sequences between the bta-miR-377 and CART 3'-UTR were highly conserved in different species.Lower sequence conservation in the miR-377 seed region occurred between cows and rats.Both of bta-miR-377 and CART were detected in bovine hypothalamus.Overexpressing bta-miR-377 mimics could significantly down-regulated the expression of CART gene mRNA and CART protein (P<0.01 or P<0.05).There was a negative correlation between bta-miR-377 and CART expression.【Conclusion】 Both bta-miR-377 and CART were expressed in bovine hypothalamic and bta-miR-377 inhibits CART expression at level of mRNA by specifically binding to CART 3'-UTR in PC12 cells.

Key words: cow; hypothalamus; CART; bta-miR-377

中图分类号:

S823.2

任静, 郝琴琴, 成俊丽, 朱芷葳, 许冬梅, 贾雪纯, 李鹏飞. bta-miR-377靶向调控牛下丘脑CART基因表达的研究[J]. 中国畜牧兽医, 2022, 49(9): 3301-3309.

REN Jing, HAO Qinqin, CHENG Junli, ZHU Zhiwei, XU Dongmei, JIA Xuechun, LI Pengfei. Expression Analysis of bta-miR-377 Regulate CART Gene in Bovine Hypothalamus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3301-3309.

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