lncRNA-6617调控猪肌内前体脂肪细胞分化的筛选与功能研究

doi:10.11843/j.issn.0366-6964.2022.06.006

摘要/Abstract

摘要： 旨在探究lncRNA-6617的生物学特性及其对猪肌内前体脂肪细胞分化的影响，以及初步探究其上游调节机制，为后续深入研究猪肌内脂肪沉积调控机制提供参考依据。本研究以饲养在相同条件下的1、90、180日龄健康马身公猪(n=3)和大白公猪(n=3)为研究对象，利用RNA-seq技术筛选脂肪型猪(马身猪)和瘦肉型猪(大白猪)肌肉特异表达的lncRNAs，并通过生物信息学分析、RT-PCR及Sanger测序鉴定lncRNA-6617生物学特性及其猪组织中的表达特征。在猪肌内前体脂肪细胞中干扰lncRNA-6617，检测lncRNA-6617对肌内脂肪细胞分化能力的影响。随后，检测m⁶A修饰相关酶在不同品种猪中的表达差异，研究lncRNA-6617的上游调控机制。结果显示，lncRNA-6617位于猪18号染色体，RAMP3基因反义链的第3内含子区域，无蛋白编码能力，主要分布于细胞核; lncRNA-6617在猪所检测各组织中均有表达，背部皮下脂肪中表达量最高，且在马身猪肌肉组织中的表达极显著高于大白猪(P < 0.01);在猪肌内前体脂肪细胞分化过程中，lncRNA-6617表达量呈上升趋势，且在分化第5天表达量达到最高; 干扰lncRNA-6617诱导分化7 d后，分化的成熟脂肪细胞明显减少，成脂关键基因CEBPα、PPARγ和aP2的表达均极显著下调(P < 0.01)。m⁶A修饰的去甲基化酶FTO在马身猪背最长肌和股二头肌中的表达极显著高于大白猪(P < 0.01)，在腰大肌中的表达显著高于大白猪(P < 0.05)，上游调节因子ZNF217在马身猪肌肉组织中的表达极显著高于大白猪(P < 0.01)，与lncRNA-6617表达模式一致。干扰FTO后，lncRNA-6617的表达极显著下调(P < 0.01)。本研究筛选了在马身猪肌肉组织中显著高表达的lncRNA-6617，并深入研究了lncRNA-6617促进猪肌内前体脂肪细胞分化的功能及其上游调节机制，丰富了脂肪细胞生成的表观调控网络。

关键词: 马身猪, lncRNA-6617, m⁶A, 肌内脂肪细胞, 成脂分化

Abstract: The aim of this study was to investigate the biological characteristics of lncRNA-6617 and its effect on the differentiation of porcine intramuscular preadipocytes, preliminarily explore its upstream regulatory mechanism, and to provide the base for further clarifying the regulation mechanism of intramuscular fat deposition in pigs. In this study, healthy Mashen boars (n=3) and Large White boars (n=3) of 1, 90, and 180 days of age were raised under the same conditions. RNA-seq technology was used to screen muscle-specific lncRNAs in fatty-type pigs (Mashen pigs) and lean-type pigs (Large White pigs). Bioinformatics analysis, RT-PCR and Sanger sequencing were used to identify lncRNA-6617 biological characteristics and expression characteristics in pig tissues. The effect of lncRNA-6617 on the differentiation ability of intramuscular adipocytes were detected after interfering with lncRNA-6617 in porcine intramuscular preadipocytes. Subsequently, the expression differences of m⁶A modification-related enzymes in different breeds of pigs were detected, and the upstream regulatory mechanism of lncRNA-6617 was studied. The results showed that lncRNA-6617 was located on pig chromosome 18, the third intron region of the RAMP3 gene antisense strand, had no protein coding ability, and mainly distributed in the nucleus. lncRNA-6617 was expressed in all detected pig tissues, with the highest expression in back subcutaneous fat, and its expression was significantly higher in MS pigs muscle tissue than that in LW pigs(P < 0.01). In the differentiation process of porcine intramuscular preadipocytes, the expression level of lncRNA-6617 showed an upward trend and reached the peak on day 5. After interfering with lncRNA-6617, the number of differentiated mature adipocytes were significantly reduced, and the expression of key adipogenic genes CEBPα, PPARγ and aP2 were significantly down-regulated (P < 0.01) on day 7 of differentiation. The expression of m⁶A-modified demethylase FTO were significantly higher in the longissimus dorsi and biceps femoris muscle of MS pigs than that of LW pigs (P < 0.01), and were significantly higher than that of LW pigs in the psoas muscle (P < 0.05). And the expression of upstream regulator ZNF217 were significantly higher in the muscle tissue of MS pigs than that in LW pigs (P < 0.01), which was consistent with the expression pattern of lncRNA-6617. After interfering with FTO, the expression of lncRNA-6617 was significantly down-regulated (P < 0.01). In conclusion, lncRNA-6617 highly expressed in the muscle tissue of Mashen pig was screened and identified. The function of lncRNA-6617 in promoting the differentiation of porcine intramuscular preadipocytes and its upstream regulation mechanism were further studied, which riched the epigenetic regulation network of adipogenesis.

Key words: Mashen pig, lncRNA-6617, m⁶A, intramuscular adipocytes, adipogenic differentiation

中图分类号:

S828.2

孟珊, 杨阳, 李睿霄, 姬梦婷, 张娜, 路畅, 蔡春波, 高鹏飞, 郭晓红, 曹果清, 李步高. lncRNA-6617调控猪肌内前体脂肪细胞分化的筛选与功能研究[J]. 畜牧兽医学报, 2022, 53(6): 1712-1722.

MENG Shan, YANG Yang, LI Ruixiao, JI Mengting, ZHANG Na, LU Chang, CAI Chunbo, GAO Pengfei, GUO Xiaohong, CAO Guoqing, LI Bugao. Screening and Functional Study of lncRNA-6617 Regulating Porcine Intramuscular Preadipocytes Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1712-1722.

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