摘要
旨在鉴定非编码RNA circNMT1,明确其组织和细胞的表达模式,以及探究过表达circNMT1对脂肪细胞分化的影响。本试验以30月龄中国沼泽水牛(信阳水牛,n=3)的心、肝、脾、肺、肾、背最长肌、背部皮下脂肪组织和前体脂肪细胞以及3T3-L1细胞为试验材料。通过半定量PCR和实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)技术对circNMT1进行鉴定、细胞定位并明确其时空表达模式。进一步分别将其过表达到3T3-L1和水牛前体脂肪细胞中,利用形态学方法及定量方法检测过表达后脂滴累积情况,同时采用qRT-PCR检测脂肪标志基因相对表达水平的变化。结果表明,circNMT1是真实存在且稳定表达的circRNA,在水牛前体脂肪细胞的细胞核和细胞质中均表达,且在脂肪组织和成熟的脂肪细胞中高表达(P<0.001)。功能获得性试验表明,在3T3-L1细胞和水牛脂肪细胞,circNMT1显著促进脂肪细胞的脂滴积累,并且显著提高成脂标志基因PPARG、C/EBPα和FABP4的相对表达水平(P<0.01)。circNMT1可能是水牛脂肪细胞分化的正调控因子,这为circNMT1在水牛脂肪细胞中的调节作用提供了新见解。
The aim of this research was to identify non-coding RNA,circNMT1,clarify its tissue and cells expression patterns,and explore the effect of circNMT1 overexpression on adipocyte differentiation.In the study,the heart,liver,spleen,lung,kidney,longissimus dorsi,dorsal subcutaneous adipose tissue and preadipocytes of 30-month-old Chinese swamp buffalo(Xinyang buffalo,n=3)and 3T3-L1 cells were used as experimental materials.Semiquantitative PCR and real-time quantitative PCR(qRT-PCR)were used to identify circNMT1,explore its location in the nucleus or cytoplasm of buffalo adipocytes.Relative expression level of circNMT1 in different tissues and preadipocytes of buffalo at different differentiation stages were detected by qRT-PCR.circNMT1 was overexpressed in 3T3-L1 cells and buffalo preadipocytes respectively,and the accumulation of lipid droplet was detected by morphology and quantitative method.Meanwhile,the relative expression level of adipogenic differentiation marker genes was detected by qRT-PCR.The results showed that circNMT1 was stably expressed circRNA,which was expressed in both nucleus and cytoplasm of buffalo preadipocytes.And circNMT1 was highly expressed in adipose tissue and maturation adipocytes(P<0.001).Gain-of-function experiments demonstrated that the overexpression of circNMT1 significantly promoted the accumulation of lipid droplets in 3T3-L1 cells and buffalo adipocytes,and significantly upregulated the relative expression level of adipogenic marker genes PPARG,C/EBPαand FABP4(P<0.01).These results indicated that circNMT1 might be a positive regulator for buffalo adipocytes differentiation,which would provide new insights into the regulatory roles of circNMT1 in buffalo adipocytes.
作者
冯雪
赵金辉
汪书哲
黄洁萍
魏雪锋
史远刚
马云
FENG Xue;ZHAO Jinhui;WANG Shuzhe;HUANG Jieping;WEI Xuefeng;SHI Yuangang;MA Yun(Key Laboratory of Ruminant Molecular and Cellular Breeding of Ningxia Hui Autonomous Region, School of Agriculture, Ningxia University, Yinchuan 750021, China;College of Life Sciences, Xinyang Normal University, Xinyang 464000, China;College of Animal Science and Technology, Guangxi University, Nanning 530000, China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2022年第4期1077-1088,共12页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(32072720,31672403)
自治区科技创新领军人才培养项目(2020GKLRLX02)
河南省千人计划中原科技创新领军人才项目(194200510022)
宁夏回族自治区重点研发引才专项(2019YCZX0068,2021BEF01002,2021NXZD1)。
