| 摘要: |
| 【目的】探究bta-miR-33b对牛前体脂肪细胞分化的作用，并对此过程中潜在的靶基因进行验证。【方法】分离培养秦川牛前体脂肪细胞并进行诱导分化，PCR检测bta-miR-33b在前体脂肪细胞分化过程中的时序表达。对牛前体脂肪细胞体外转染bta-miR-33b的mimic、mimic negative control (mimic NC)、inhibitor、inhibitor negative control (inhibitor NC)进行诱导分化培养，于分化培养的不同时间取样，检测脂肪细胞分化标志基因（过氧化物酶体增殖物激活受体γ（PPARγ）基因、CCAAT/增强子结合蛋白α（C/EBPα）基因、脂肪酸结合蛋白4（FABP4）基因）的mRNA表达量及甘油三酯和脂滴含量。预测bta-miR-33b的靶基因，并通过双荧光素酶报告系统验证靶向关系、Western Blot检测靶蛋白的表达量。【结果】在秦川牛前体脂肪细胞分化过程中，bta-miR-33b的表达量呈下降趋势。在前体脂肪细胞分化过程中，bta-miR-33b mimic可抑制脂肪分化标志基因的表达，降低细胞中甘油三酯和脂滴的含量；bta-miR-33b inhibitor可促进脂肪分化标志基因的表达，提高甘油三酯和脂滴的含量。高迁移率族蛋白A2(High mobility group AT-hook 2,HMGA2)基因、扭曲相关蛋白（Twist family bHLH transcription factor 1,TWIST1）基因是bta-miR-33b的靶基因；脂肪细胞分化过程中，bta-miR-33b能够调控HMGA2和TWIST1蛋白水平的表达。【结论】bta-miR-33b对秦川牛前体脂肪细胞体外分化具有抑制作用；HMGA2和TWIST1是bta-miR-33b的靶基因。 |
| 关键词: 秦川牛 bta-miR-33b 脂肪细胞分化 HMGA2 TWIST1 |
| DOI： |
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| 基金项目:国家重点研发计划项目（2018YFD0501700）；国家“863”计划项目（2013AA102505）；国家科技支撑计划项目（2015-BAD03B04）；国家肉牛牦牛产业技术体系项目（CARS-37） |
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| Inhibitory effect of bta-miR-33b on pre-adipocytesdifferentiation of cattle in vitro |
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ZHANG Wenzhen,ZHANG Song,WANG Li,et al
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| Abstract: |
| 【Objective】The present study investigated the role of bta-miR-33b on cattle pre-adipocytes and identified the potential-mediated target genes in the bioprocess.【Method】The Qinchuan cattle preadipocytes were isolated and cultured before inducting differentiation,and the expression of bta-miR-33b during differentiation was measured by RT-PCR.After transfecting mimic,mimic negative control (mimic NC),inhibitor,and inhibitor negative control (inhibitor NC) of bta-miR-33b into pre adipocytes and inducing differentiation,the expression levels of adipogenic markers including peroxisome proliferators-activated receptor γ（PPARγ）,CCAAT/enhancer binding protein α （C/EBPα） and fatty acid binding protein （FABP4） and contents of triglyceride and lipid droplet were measured in different days.The target genes of bta-miR-33b were predicted and identified by the dual-luciferase assay,and the protein levels were measured by Western Blot.【Result】The expression of bta-miR-33b gradually decreased with preadipocytes differentiation.bta-miR-33b mimic inhibited the expression of adipogenic markers and contents of triglyceride and lipid droplet.bta-miR-33 inhibitor promoted the expression of adipogenic markers and contents of triglyceride and lipid droplet.High mobility group AT-hook （HMGA2）and twist family bHLH transcription factor 1 （TWIST1） were directly target genes of bta-miR-33b.Expression of HMGA2 and TWIST1 was regulated in adipocytes differentiation.【Conclusion】bta-miR-33b inhibited pre-adipocytes differentiation of Qinchuan cattle in vitro and it had two target genes of HMGA2 and TWIST1. |
| Key words: Qinchuan cattle bta-miR-33b pre-adipocytes differentiation HMGA2 TWIST1 |
