miR-106b-5p靶向KLF4调控山羊肌内前体脂肪细胞分化

doi:10.11843/j.issn.0366-6964.2020.006

摘要/Abstract

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。

关键词: 山羊, miR-106b-5p, KLF4, 脂肪细胞, 荧光素酶活性试验

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.

Key words: goat, miR-106b-5p, KLF4, adipocyte, luciferase activity assay

中图分类号:

S827.2

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.

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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