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| miR-376b-5p调控绵羊产羔性状相关基因IGF1的表达 |
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李文韬1,2,周丽娟3,刘子嶷1,2,王鹏1,2,储明星2*,刘玉芳1,2*
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| (1. 河北工程大学 生命科学与食品工程学院, 河北 邯郸 056021;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;3.河北省沧州市东光县农业农村局, 河北 沧州 061600) |
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| 摘要: |
| 为探讨miR-376b-5p靶向调控IGF1影响绵羊多羔性状的分子机制，本研究根据产羔记录选取高、低产羔数小尾寒羊各5只，同期发情处理后，采集卵泡期卵巢及其他6个组织，通过RT-qPCR、Western blot和双荧光素酶报告基因检测系统分析miR-376b-5p与IGF1在高、低产羔数小尾寒羊各组织中的表达量；并确定二者之间的靶向关系；随后，将体外合成的 miR-376b-5p模拟物(mimic)和抑制物(inhibitor)转染至绵羊卵巢颗粒细胞中，检测miR-376b-5p对IGF1表达水平的调控情况，同时对多羔性状TGF-β信号通路中TGFβ1和PTGS2标志因子的表达进行分析。结果表明:1)组织表达谱显示，IGF1在绵羊卵巢组织中的表达量显著高于其他组织(P<0.05)；RT-qPCR和Western blot显示，IGF1在高产羔数小尾寒羊卵巢组织中的表达量极显著高于低产羔数小尾寒羊(P<0.01)；miR-376b-5p定量结果显示其表达趋势与IGF1相反(P<0.01)；2)在线软件预测和双荧光素酶报告系统检测显示，miR-376b-5p可靶向结合IGF1 3′UTR序列；3)在绵羊卵巢颗粒细胞中过表达miR-376b-5p后，IGF1蛋白和mRNA表达水平均显著下降(P<0.05)，而抑制miR-376b-5p后则相反，表明miR-376b-5p可显著抑制IGF1的表达；4)在绵羊卵巢颗粒细胞中过表达miR-376b-5p后，TGF-β信号通路中TGFβ1、PTGS2的表达量均显著低于阴性对照组(P<0.05)。综上所述，在绵羊卵巢颗粒细胞中miR-376b-5p靶向结合IGF1 3′UTR区域，并通过抑制IGF1和TGF-β信号通路调控颗粒细胞增殖及绵羊繁殖，为进一步揭示绵羊多羔性状形成的分子机制及分子育种提供理论基础。 |
| 关键词: miR-376b-5p IGF1 绵羊 卵巢 |
| DOI：10.11841/j.issn.1007-4333.2023.04.10 |
| 投稿时间：2022-08-16 |
| 基金项目:国家自然科学基金项目(32172704)；财政部和农业农村部国家现代农业产业技术体系资助(CARS-38)；中国农业科学院科技创新工程(CAAS-ZDRW202106；ASTIP-IAS13) |
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| miR-376b-5p regulated the expression of lambing trait related gene IGF1 in sheep |
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LI Wentao1,2,ZHOU Lijuan3,LIU Ziyi1,2,WANG Peng1,2,CHU Mingxing2*,LIU Yufang1,2*
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| (1.College of Life Sciences and Food Engineering, Hebei Engineering University, Handan 056021, China;2.Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;3.Agricultural and Rural Bureau of Dongguang, Cangzhou 061600, China) |
| Abstract: |
| In order to explore the molecular mechanism of miR-376b-5p targeting and regulating IGF1 to influence the high-fertility lambing trait in sheep, five small-tailed Han sheep with high and low lambs were selected according to the lambing records. Ovarians and other six tissues were collected at follicular phase after simultaneous oestrus treatment. The expression of miR-376b-5p and IGF1 in ovarian tissues of high- and low-fertility sheep were analyzed by RT-qPCR, Western blot and dual luciferase reporter assay. And then the targeting relationship between miR-376b-5p and IGF1 was determined. Subsequently, the miR-376b-5p mimic and inhibitor were transfected into sheep primary granulosa cells in vitro to detect the expression of IGF1, and the expression of TGFβ1 and PTGS2, markers of the TGF-β signaling pathway, a pathway related to high-fertility laming trait, was also analyzed. The results showed that: 1)Tissues expression profiles showed that IGF1 was significantly higher in ovarian tissues of sheep than that in other tissues(P<0. 05). RT-qPCR and Western blot results showed that the expression of IGF1 in ovarian tissues of high-fertility sheep were significantly higher than that of low-fertility sheep(P<0. 01). The RT-qPCR results of miR-376b-5p showed that its expression trend was opposite to that of IGF1(P<0. 01). 2)The result of online software and dual luciferase assay showed that the miR-376b-5p could target binding to IGF1 3′UTR sequence. 3)IGF1 protein and mRNA expression levels were significantly decreased after overexpression of miR-376b-5p in sheep ovarian granulosa cells(P<0. 05), while the opposite was true after inhibition of miR-376b-5p, indicating that miR-376b-5p significantly inhibited IGF1 expression. 4)The expression of TGFβ1 and PTGS2 in the TGF-β signaling pathway was significantly lower than that of the negative control group after overexpression of miR-376b-5p in sheep ovarian granulosa cells(P<0. 05). In conclusion, miR-376b-5p targets the IGF1 3′UTR region in ovarian granulosa cells of sheep, and regulates granulosa cell proliferation and sheep reproduction by inhibiting IGF1 and TGF-β signaling pathways, providing a theoretical basis for further unraveling the molecular mechanism of sheep high-fertility lambing trait formation and molecular breeding. |
| Key words: miR-376b-5p IGF1 sheep ovary |
