LRTN4RL1-AS通过miR-27a-3p靶向PPARγ调节奶牛乳腺细胞乳脂合成  

LRTN4RL1-AS Mediates Milk Fat Synthesis in Bovine Mammary Epithelial Cells through miR-27a-3p Targeting PPARγ

作　　者：贾鸿儒 杨超群 王萌[1] 吴章情 昝林森[1] 杨武才[1] JIA Hongru;YANG Chaoqun;WANG Meng;WU Zhangqing;ZAN Linsen;YANG Wucai(National Beef Cattle Improvement Center,College of Animal Science and Technology,Northwest A&F University,Yangling 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,杨凌712100

出　　处：《畜牧兽医学报》2023年第4期1465-1477,共13页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：陕西省自然科学基金(2021JM-100)。

摘　　要：旨在探究lncRNA-LRTN4RL1-AS/miR-27a-3p/PPARγ轴在奶牛乳脂合成中的作用及其调控机制。本研究以奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)为试验对象,对处理组与对照组进行3个生物学重复,通过实时荧光定量(real time quantitative PCR,RT-qPCR)、WB(Western blot)、油红O染色和甘油三酯检测、双荧光素酶报告等技术检测lncRNA-LRTN4RL1-AS对BMECs中甘油三酯积累及脂质合成相关基因表达量的影响,验证LRTN4RL1-AS-miR-27a-3p-PPARγ存在内源竞争RNA(competing endogenous RNAs,ceRNA)的靶向关系。结果表明,LRTN4RL1-AS主要在细胞质中表达,干扰LRTN4RL1-AS后显著抑制了BMECs的脂滴形成和甘油三酯积累(P<0.05),显著下调脂质合成相关基因CEBPα(P<0.01)、CEBPβ(P<0.01)和PPARγ(P<0.05)表达,且显著上调脂质分解基因HSL表达(P<0.05);双荧光素酶报告试验发现,转染mimics-miR-27a-3p,LRTN4RL1-AS突变型组荧光素酶活性显著高于LRTN4RL1-AS野生型组(P<0.01),证实LRTN4RL1-AS与miR-27a-3p之间存在靶向结合关系;共转染试验发现,inhibitor-miR-27a-3p逆转了干扰LRTN4RL1-AS对乳脂合成的抑制作用(P<0.05),同时降低了si-LRTN4RL1-AS对PPARγ基因的表达抑制。结果显示,LRTN4RL1-AS通过竞争性结合miR-27a-3p调控PPARγ表达来调节BMECs乳脂合成。This study aimed to explore the role and regulatory mechanism of lncRNA-LRTN4RL1-AS/miR-27a-3p/PPARγaxis in milk fat synthesis of dairy cows.Bovine mammary epithelial cells(BMECs)were used as experimental objects.Three biological repeats were carried out for the treatment group and the control group.Real time quantitative PCR(RT-qPCR),Western blot(WB),Oil Red O staining,triglyceride detection and double luciferase report were used to detect the effect of lncRNA-LRTN4RL1-AS on the expression of triglyceride accumulation and lipid synthesis related genes in BMECs.LRTN4RL1-AS-miR-27a-3p-PPARγhad targeting relationship of competing endogenous RNAs(ceRNA)was verified.The results showed that LRTN4RL1-AS was mainly expressed in the cytoplasm.After interfering with LRTN4RL1-AS,the lipid droplet formation and triglyceride accumulation of BMECs were significantly inhibited(P<0.05),and the expression of lipid synthesis related genes CEBPα(P<0.01),CEBPβ(P<0.01)and PPARγwere significantly down-regulated(P<0.05),and the expression of lipid catabolism gene HSL was significantly up-regulated(P<0.05).Double luciferase report assay showed that the luciferase activity in mimics-miR-27a-3p in LRTN4RL1-AS mutant group was significantly higher than that in LRTN4RL1-AS wild type group(P<0.01).It was confirmed there was a targeted binding relationship between LRTN4RL1-AS and bta-miR-27a-3p.Cotransfection experiment result showed that inhibitor-miR-27a-3p reversed the inhibition effect of interference LRTN4RL1-AS on milk fat synthesis(P<0.05),and reduced the inhibition effect of si-LRTN4RL1-AS on PPARγgene expression.The results show that LRTN4RL1-AS regulates milk fat synthesis of BMECs by competitively binding miR-27a-3p to regulate the expression of PPARγ.

关 键 词：乳脂肪 lncRNA PPARΓ miR-27a-3p 

分 类 号：S823.91[农业科学—畜牧学]