| 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。 |
| 关键词: 绵羊 miR-370-3p COL4A3 甲状腺 高繁殖力 |
| DOI：10.11841/j.issn.1007-4333.2023.09.10 |
| 投稿时间：2022-11-08 |
| 基金项目: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) |
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| Preliminary study on the regulation of fertility traits by miR-370-3p targeting COL4A3 gene in sheep thyroid gland |
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CHANG Cheng1,2,ZHANG Qian1,HE Xiaoyun2,CHU Mingxing2*,LIANG Chen1*
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| (1.College of Animal Science, Shanxi Agricultural University, Taigu, 030801, China;2.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) |
| Abstract: |
| 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. |
| Key words: sheep miR-370-3p COL4A3 thyroid gland high fertility |
