基于Solexa测序筛选的miRNAs在猪背膘和肌内脂肪细胞分化中的差异性调控机制研究

Preadipocytes derived from backfat of pigs differs significantly from intramuscular tissue when differentiate into mature adipocytes, miRNA regulates this process through Wnt signaling but the underlying mechanism remains to be investigated. Elucidating such mechanisms is of great importance. Previous research conducted with 5 months-old Yorkshire and Meishan pigs employing Solexa sequencing revealed that miR-135, miR-183 and miR-215 were differentially expressed and were predicted to targeted towards APC, LRP6 and Tcf7 of Wnt signaling. The present study determines expression profile of the 3 miRNA with RT-PCR; establishes pig backfat and intramuscular fat preadipocyte cell line; by means of cell transfection, Northern blot, q-PCR and western blot investigates the differential effect of the 3 miRNA on preadipocyte differentiation, by employing luciferase reporting system and overexpression of target genes verifies effect of miRNA on Wnt target genes and elucidates their differential regulating mechanism on backfat and intramuscular fat development. The present study provides new theoretical basis for intramuscular fat development and pathogenesis and control of adipogenic differentiation related diseases.

猪背膘组织和肌内脂肪组织的前脂肪细胞在分化中有显著差异性，miRNA经Wnt信号通路影响了脂肪细胞分化，但机制仍不清楚。阐明miRNA在其中的调控机制具有重要意义。我们前期研究已对5月龄大白猪和梅山猪背膘脂肪组织进行Solexa测序，并筛选出差异表达miR-135、miR-183和miR-215，预测其分别靶向wnt通路的APC、LRP6和Tcf7基因。本项目将用RT-PCR方法研究3个miRNA时空表达谱；建立猪背膘脂肪和肌内脂肪组织的前脂肪细胞系；用细胞转染、Northern blot、q-PCR和Western blot等技术，研究3个miRNA对两种前脂肪细胞分化的影响和差异性；用双荧光素酶和超表达靶基因等证明miRNA对Wnt通路靶基因的影响，并阐明它们对背膘和肌内脂肪形成的差异性调控机制。为解析肌内脂肪性状的形成、揭示与脂肪细胞分化发育相关的发病机制和控制措施提供新的理论依据。

猪皮下脂肪和肌内脂肪组织的前脂肪细胞在分化中有显著差异性，miRNA 经Wnt信号通路影响了脂肪细胞分化，但机制仍不清楚。本项目研究发现miR-135、miR-183和miR-215在大白猪3个不同生长阶段的不同组织中的表达各有差异，表明miRNAs对脂肪细胞分化与脂肪沉积的调控具有时空差异。采用多种实验技术证实miR-135、miR-183和miR-215通过LRP6、FNDC3B、APC、CTNNBIP1、ELOVL2和ELOVL6等靶基因参与脂肪生成的重要开关Wnt/β-catenin信号通路调控脂肪细胞分化；miR-146b通过抑制TGFβ信号通路中的Smad4的表达，抑制TGFβ通路下游的基因CTGF的表达从而促进脂肪细胞的分化；miR-224能够抑制EGR2的表达，可能通过抑制C/EBPβ的表达抑制脂肪的分化；miR-224通过靶向ACSL4参与调控脂肪酸代谢。以上研究结果证实了脂肪细胞分化和脂肪形成的调控机制具有差异性。同时，研究发现miR-135a-1和miR-135a-2都是基因内miRNA，但并不与宿主基因共转录，而是具有独立转录机制；STAT5a同时结合miR-135a-1和miR-135a-2的核心启动子从而促进成熟miR-135a的表达；STAT5a与miR-135a的靶基因APC在脂肪细胞分化过程中存在一个潜在的负反馈调节环路。本项目所获取的试验结果将为解析肌内脂肪性状的形成、揭示与脂肪细胞分化发育相关的发病机制和控制措施提供新的理论依据。在Cell Signal、Int J Biochem Cell Biol、Journal of Molecular Endocrinolog等期刊发表密切相关SCI收录论文6篇。