miR-128a调控猪前体脂肪细胞分化及脂肪代谢的分子机制研究

miRNA influences the process of preadipocyte differentiation and fat metabolism in pigs, but the underlying mechanism remains to be investigated. The backfat tissues of Yorkshire and Meishan pigs were collected at five months after birth for miRNA-based Solexa sequencing, the results revealed that miR-128a was differentially expressed between two samples, and SERTAD2 was predicted to be the target of miR-128a by serveral softwares. The present study will investigate the expression profile and pattern of miR-128a by qPCR. Then the porcine preadipocyte cell line from backfat tissue will be established for elucidating the role of miR-128a in the process of preadipocyte differentiation and fat metabolism by cell transfection and induction, qPCR, Northern blot and Western blot. The target effect between miR-128a and SERTAD2 and the regulatory effect of miR-128a on transcriptional activity of genes downstream the E2F-1/DP-1 complex will be further determined by using the dual luciferase reporter system, over-expression and knockdown of target gene, and construction of eukaryotic expression vector. And the mechanism that miR-128a regulats porcine preadipocyte differentiation and fat metabolism will be clearly illuminated. The results from this study will provide more insight into the mechanism of porcine adipocyte differentiation and development from the standpoint of miRNA, and also put forward the new theoretical basis for regulation of fat metabolism.

miRNA影响猪的前体脂肪细胞分化及脂肪代谢进程，但其介导的调控机制仍不清楚。前期研究对5月龄大白猪和梅山猪背部脂肪组织的miRNA进行Solexa测序，筛选出差异表达的miR-128a，预测其靶基因为SERTAD2。本项目采用qPCR方法研究miR-128a的表达谱及其表达规律；建立猪背部脂肪组织的前体脂肪细胞系，通过细胞转染诱导、qPCR、Northern blot和Western blot等技术研究miR-128a对猪前体脂肪细胞分化及脂肪代谢的影响；利用双荧光素酶报告系统、靶基因的超表达和敲减以及真核表达载体构建等技术证明miR-128a的靶基因效应及其对E2F-1/DP-1复合物下游基因转录活性的调控作用，阐明miR-128a调控猪前体脂肪细胞分化及脂肪代谢的分子机制。本项目若被证实，将从miRNA角度解析猪脂肪细胞分化及发育相关机制，为脂肪代谢调节提供新的理论依据。

miRNA是一类内源性的小分子非编码RNA，其在脂肪形成和脂质代谢过程中起着重要的调控作用。本课题组的前期研究表明，miR-128-3p在瘦肉型大白猪背脂中的表达量是脂肪型梅山猪的9.86倍，也有文献报道蓝塘猪背最长肌中miR-128-3p的表达量是皮下脂肪组织中的2.77倍。这些结果暗示着miR-128-3p可能参与脂肪形成和脂质代谢，但目前关于miR-128-3p对脂肪细胞分化、脂肪形成相关的作用研究还未见报道。. 本研究以小鼠3T3-L1前体脂肪细胞、成熟脂肪细胞为研究对象，结果发现：（1）随着前体脂肪细胞的分化，miR-128-3p的表达量显著降低。（2）在细胞分化过程中，超表达miR-128-3p显著抑制脂肪形成标记基因（C/EBPα、PPARγ、FAS、ap2）表达、脂滴积累和甘油三酯含量。（3）miR-128-3p通过增加前体脂肪细胞G0/G1期比例、降低G2/M期比例，从而阻滞细胞周期，进而抑制前体脂肪细胞的增殖。（4）双荧光素酶报告基因、QPCR和蛋白印迹试验显示，PPARγ和SERTAD2是miR-128-3p的靶基因。（5）成熟3T3-L1脂肪细胞中，miR-128-3p可显著提高脂解关键基因ATGL和HSL的表达和游离脂肪酸含量，从而促进甘油三酯分解。（6）敲减SERTAD2基因，抑制成熟脂肪细胞中脂滴积累和甘油三酯含量，进而抑制脂肪形成。其中，成脂基因（C/EBPα、PPARγ）的表达不受影响，而脂解基因（ATGL、HSL）的表达和游离脂肪酸含量显著升高。. 本研究表明，miR-128-3p通过靶向PPARγ基因，抑制脂肪形成标记基因C/EBPα、FAS、ap2的表达，阻碍3T3-L1前体脂肪细胞分化，导致甘油三酯的积累受阻。另一方面，miR-128-3p通过靶向抑制SERTAD2，促进脂解基因ATGL和HSL的表达，从而激活甘油三酯分解和脂肪水解进程，进而抑制脂肪形成。本研究为深入解析miRNA介导调控脂肪形成和脂质代谢的分子机制提供了理论依据。