| miR-106b-5p靶向KLF4调控山羊肌内前体脂肪细胞分化 |
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| 引用本文： | 杜宇,赵越,林亚秋,朱江江,王永,马洁琼,谢光杰.miR-106b-5p靶向KLF4调控山羊肌内前体脂肪细胞分化[J].畜牧兽医学报,2020,51(6):1219-1228. |
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| 作者姓名： | 杜宇 赵越 林亚秋 朱江江 王永 马洁琼 谢光杰 |
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| 作者单位： | 1. 西南民族大学 青藏高原动物遗传资源保护与利用教育部/四川省重点实验室, 成都 610041;2. 西南民族大学 生命科学与技术学院, 成都 610041 |
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| 基金项目： | 国家重点研究计划专项（2018YFD0502002）；国家自然科学基金（31672395）；四川省应用基础研究计划重点项目（2018JY0036） |
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| 摘 要： | 旨在明确miR-106b-5p对山羊肌内前体脂肪细胞分化的影响,并确定这种作用是通过靶向KLF4来实现的。本研究利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术检测miR-106b-5p在山羊肌内前体脂肪细胞分化过程中的表达模式,通过脂质体转染技术将miR-106b-5p mimic和miR-106b-5p inhibitor转入体外培养的山羊肌内前体脂肪细胞,油红O染色法从形态学验证miR-106b-5p对脂肪细胞中脂滴积聚的影响,qRT-PCR检测预测的靶标基因KLF4和脂肪分化标志基因的表达情况,利用双荧光素酶报告系统鉴定miR-106b-5p与KLF4的靶标关系。qRT-PCR结果显示,miR-106b-5p在山羊肌内前体脂肪细胞诱导分化第3天时表达量最高。在山羊肌内脂肪细胞中干扰miR-106b-5p后油红O染色显示脂滴聚积减少,过表达miR-106b-5p后脂滴聚积增加。在山羊肌内前体脂肪细胞中转染miR-106b-5p inhibitor后PPARγ表达量显著降低(P<0.05),而KLF4表达量极显著升高(P<0.01);转染miR-106b-5p mimic后LPL和PPARγ表达量极显著升高(P<0.01)。荧光素酶活性试验结果显示,过表达miR-106b-5p可显著抑制KLF4荧光活性。miR-106b-5p通过靶向并负调节KLF4的表达促进山羊肌内脂肪细胞分化。
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| 关 键 词： | 山羊 miR-106b-5p KLF4 脂肪细胞 荧光素酶活性试验 |
| 收稿时间： | 2019-11-07 |
miR-106b-5p Regulates the Differentiation of Goat Intramuscular Preadipocytes by Targeting KLF4 |
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| DU Yu,ZHAO Yue,LIN Yaqiu,ZHU Jiangjiang,WANG Yong,MA Jieqiong,XIE Guangjie.miR-106b-5p Regulates the Differentiation of Goat Intramuscular Preadipocytes by Targeting KLF4[J].Acta Veterinaria et Zootechnica Sinica,2020,51(6):1219-1228. |
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| Authors: | DU Yu ZHAO Yue LIN Yaqiu ZHU Jiangjiang WANG Yong MA Jieqiong XIE Guangjie |
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| Institution: | 1. Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Protection and Utilization of Ministry of Education/Sichuan Province, Southwest Minzu University, Chengdu 610041, China;2. College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China |
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| Abstract: | This study aimed to explore the effect of miR-106b-5p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-106b-5p played its roles via targeting KLF4. The quantitative real-time PCR (qRT-PCR) was used to detect the expression pattern of miR-106b-5p during the differentiation of goat intramuscular preadipocytes. The miR-106b-5p mimic and miR-106b-5p inhibitor were transfected into goat intramuscular preadipocytes cultured in vitro by liposome transfection. The effect of miR-106b-5p on lipid droplet accumulation was verified using the Oil Red O Staining. The expression levels of predicted target gene KLF4 targeting miR-106b-5p and adipogenic marker genes were detected using qRT-PCR. The target relationship between miR-106b-5p and KLF4 was detected by dual luciferase reporter system. The result of qRT-PCR showed that the highest expression level of miR-106b-5p was at day 3 of induced differentiation of goat intramuscular preadipocytes. The Oil Red O staining result showed that lipid droplet accumulation was decreased after treated by miR-106b-5p inhibitor, however, it had an opposite result after treated by miR-106b-5p mimic in goat intramuscular adipocytes. The expression level of PPARγ was significantly decreased (P<0.05) and the expression level of KLF4 was extremely significantly increased (P<0.01) in the miR-106b-5p inhibitor treatment. The expression levels of LPL and PPARγ were extremely significantly increased (P<0.01) in the miR-106b-5p mimic treatment. The luciferase activity assay result showed that luciferase activity of KLF4 was significantly inhibited in the miR-106b-5p mimic treatment. miR-106b-5p promotes the differentiation of goat intramuscular preadipocytes via targeting and negatively regulating the expression of KLF4. |
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| Keywords: | goat miR-106b-5p KLF4 adipocyte luciferase activity assay |
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