绵羊甲状腺miR-370-3p靶向COL4A3基因调控繁殖力性状的初步研究  

Preliminary study on the regulation of fertility traits by miR-370-3p targeting COL4A3 gene in sheep thyroid gland

作　　者：常成 张茜 贺小云[2] 储明星[2] 梁琛[1] CHANG Cheng;ZHANG Qian;HE Xiaoyun;CHU Mingxing;LIANG Chen(College of Animal Science,Shanxi Agricultural University,Taigu,030801,China;Key Laboratory of Animal Genetics,Breeding and Reproduction of Ministry of Agriculture and Rural Affairs/Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]山西农业大学动物科学学院,山西太谷030801 [2]中国农业科学院北京畜牧兽医研究所/农业农村部动物遗传育种与繁殖重点实验室,北京100193

出　　处：《中国农业大学学报》2023年第9期108-116,共9页Journal of China Agricultural University

基　　金：国家自然科学基金项目(32172704);中央级公益性科研院所基本科研业务费专项(2021-YWF-ZYSQ-14);财政部农业农村部国家肉羊产业技术体系(CARS-38);中国农业科学院科技创新工程(ASTIP-IAS13);山西省基础研究计划面上项目(20210302123371);山西农业大学博士科研启动项目(2021BQ04)。

摘　　要：为探讨小尾寒羊甲状腺组织中与繁殖功能相关的miR-370-3p与COL4A3的靶向关系,本研究利用miRDB与Targetscan网站预测了miR-370-3p的靶基因,并取其交集进行功能富集分析,通过RNAhybrid与Targetscan网站预测绵羊miR-370-3p与候选靶基因的结合位点;随后检测了miR-370-3p与COL4A3基因在小尾寒羊甲状腺组织中的相对表达量。构建psiCHECK2-COL4A3-3′UTR-野生型/突变型双荧光素酶载体,并将其与miR-370-3p mimics/mimics NC共转染至HEK293T细胞并检测荧光活性。结果显示:1)miR-370-3p的靶基因可富集到孕酮介导的卵母细胞成熟、卵母细胞减数分裂、HIF-1信号通路、MAPK信号通路、mTOR信号通路、Ras信号通路和FoxO信号通路等与动物繁殖有关的信号通路中。2)miR-370-3p可与COL4A3基因3′UTR区域结合,且两者在小尾寒羊甲状腺组织中表达呈相反趋势。3)共转染psiCHECK2-COL4A3-3′UTR-野生型和miR-370-3p mimics组的荧光素酶活性显著低于共转染psiCHECK2-COL4A3-3′UTR-野生型和miR-370-3p mimics NC组、共转染psiCHECK2-COL4A3-3′UTR-突变型和miR-370-3p mimics组、共转染psiCHECK2-COL4A3-3′UTR-突变型和miR-370-3p mimics NC组(P<0.05),而这三组间无显著差异(P>0.05)。综上,miR-370-3p与COL4A3存在靶向关系,且可靶向结合COL4A3-3′UTR区域,为进一步探究miR-370-3p影响绵羊甲状腺功能与绵羊繁殖力的分子机制提供了依据。To explore the potential function of miR-370-3p associated with reproductive function and its targeting relationship with COL4A3 in the thyroid tissue of Small Tail Han(STH)sheep,this study predicted the target genes of miR-370-3p using the miRDB website and Targetscan website,and took their intersections for functional enrichment analysis,and the binding site of sheep miR-370-3p to candidate target genes were predicted by the RNAhybrid and Targetscan website.Subsequently,the relative expression levels of miR-370-3p and COL4A3 in the thyroid tissue of STH sheep were detected.The psiCHECK2-COL4A3-3′UTR wild-type(WT)/mutant-type(MT)Dual-luciferase reporter vectors were constructed,and co-transfected with miR-370-3p mimics/mimics NC into HEK293T cells to detect fluorescence activity.The results showed that:1)The target genes of miR-370-3p were enriched in progesterone-mediated oocyte maturation,oocyte meiosis,HIF-1 signaling pathway,MAPK signaling pathway,mTOR signaling pathway,Ras signaling pathway,FoxO signaling pathway,and other signaling pathways related to animal reproduction.2)RT-qPCR results showed that the expression of miR-370-3p and COL4A3 in the thyroid tissue of STH sheep showed an opposite trend.3)The luciferase activities of co-transfected psiCHECK2-COL4A3-3′UTR-WT and miR-370-3p mimics were significantly lower than co-transfected psiCHECK2-COL4A3-3′UTR-WT and miR-370-3p NC mimics group,co-transfected psiCHECK2-COL4A3-3′UTR-MT and miR-370-3p mimics group,co-transfected psiCHECK2-COL4A3-3′UTR-MT and miR-370-3p mimics NC group(P<0.05).There was no significant difference among the other three groups(P>0.05).The above results indicated that miR-370-3p targets and binds COL4A3-3′UTR and inhibits its transcriptional activity,suggesting that miR-370-3p can target binding to COL4A3-3′UTR region,providing a basis for further studies on the molecular mechanism of miR-370-3p affecting sheep thyroid function and sheep fertility.

关 键 词：绵羊 miR-370-3p COL4A3 甲状腺 高繁殖力 

分 类 号：S826[农业科学—畜牧学]