bta-miR-33b对牛前体脂肪细胞体外分化的抑制作用研究  

Inhibitory effect of bta-miR-33b on pre-adipocytes differentiation of cattle in vitro

作　　者：张文振 张松[1] 王力[1] 王晓宇[1] Rajwali Khan 成功[1] 昝林森[1] ZHANG Wenzhen;ZHANG Song;WANG Li;WANG Xiaoyu;Rajwali Khan;CHENG Gong;ZAN Linsen(College of Animal Science and Technology,Northwest A&F University/National Beef Cattle Improvement Center,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学动物科技学院/国家肉牛改良中心,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2020年第4期1-9,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：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)。

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。【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 receptorγ(PPARγ),CCAAT/enhancer binding proteinα(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.

关 键 词：秦川牛 bta-miR-33b 脂肪细胞分化 HMGA2 TWIST1 

分 类 号：S823[农业科学—畜牧学] Q343[农业科学—畜牧兽医]