MYOZ2通过负调控TCAP的表达促进C3H10T1/2细胞成脂分化

本研究旨在明确钙调磷酸酶肌小节结合蛋白2（myozenin2，MYOZ2）的组织表达特性，阐明其对C3H10T1/2细胞成脂分化的影响及可能的作用机制。采集180日龄马身猪、60日龄ICR小鼠、35日龄罗斯肉鸡和12月龄小尾寒羊背最长肌、皮下脂肪和肝组织，检测MYOZ2基因mRNA表达。结果显示，MYOZ2基因在所检测的物种中表现出相似的组织表达规律，在背最长肌表达量最高，皮下脂肪与肝组织低水平表达。在C3H10T1/2细胞中沉默MYOZ2基因后，qRT-PCR结果显示，与对照组相比，成脂关键基因PPARγ、FABP4表达极显著下调（P<0.01）；Western 印迹结果表明，PPARγ蛋白含量显著降低（P<0.05）；油红O染色发现，沉默MYOZ2基因后脂滴数明显减少，甘油三酯含量显著降低（P<0.05）。利用qRT-PCR技术检测沉默MYOZ2基因后，脂肪代谢相关基因SCD、FASN、SREBP1、NR1H3、DGAT1、PNPLA2、HSL、CES1、CPT1等的表达，结果显示，SCD、FASN、SREBP1、PNPLA2、HSL极显著下调（P<0.01），NR1H3显著下调（P<0.05），DGAT1表达下调但差异不显著，CES1、CPT1显著上调（P<0.05）。利用STRING数据库构建MYOZ2相关蛋白质互作网络图，发现MYOZ2可能通过肌联蛋白帽（Titin-cap，TCAP）与PPARγ相互作用进而影响成脂分化。沉默TCAP基因，qRT-PCR结果显示，与对照组相比，成脂关键基因PPARγ、FABP4表达极显著上调（P<0.01）；Western 印迹结果表明，与对照组相比，PPARγ蛋白含量显著升高（P<0.05）；油红O染色发现，沉默TCAP基因后脂滴数明显增多，甘油三酯含量显著升高（P<0.05）。利用qRT-PCR技术检测沉默MYOZ2后TCAP基因的表达结果显示，沉默MYOZ2后TCAP的表达极显著升高（P<0.01）。综上所述，MYOZ2基因在背最长肌中表达量最高，在皮下脂肪和肝组织中也有少量表达。此外，MYOZ2可能通过MYOZ2-TCAP-PPARγ途径影响成脂关键基因PPARγ和FABP4的表达，进而调控脂肪酸代谢相关基因SCD、FASN、SREBP1、NR1H3、DGAT1、PNPLA2、HSL、CES1、CPT1，在成脂分化过程中发挥重要作用。

MYOZ2 Promoted Adipogenic Differentiation of C3H10T1/2 Cells by Negatively Regulating the Expression of TCAP

The objective of this study was to investigate the expression profile of the myozenin2 (MYOZ2) gene and elucidate its effect on adipogenic differentiation of C3H10T1/2 cells and its possible mechanism. The longissimus dorsi, subcutaneous fat and liver tissue was collected from 180-day-old Mashen pigs, 60-day-old ICR mice, 35-day-old Ross broiler and 12-month-old Small tail han sheep, and the expression profile of the MYOZ2 gene mRNA was detected. The results showed that the MYOZ2 gene has similar patterns of tissue expression in examined species, with the highest expression level in longissimus dorsi, and a small amount of expression in the subcutaneous fat and liver tissue. After the MYOZ2 gene was silenced in C3H10T1/2 cells, qRT-PCR results showed that the expression levels of key adipogenic genes PPARγ and FABP4 were significantly down-regulated compared with the control group (P<0.01); Western Blotting results showed that the PPARγ protein content was significantly decreased (P<0.05); Oil red O staining showed that the number of lipid droplets and the content of triglyceride were significantly decreased after silencing MYOZ2 (P<0.05). The expression of fatty acid metabolism related genes SCD, FASN, SREBP1, NR1H3, DGAT1, PNPLA2, HSL, CES1, CPT1 after MYOZ2 silencing were detected by qRT-PCR. The results showed that SCD, FASN, SREBP1, PNPLA2 and HSL were significantly down-regulated (P<0.01), NR1H3 was significantly reduced (P<0.05), DGAT1 expression was down-regulated but the difference was not significant, CES1 and CPT1 were significantly up-regulated (P<0.05). The STRING database was used to construct a MYOZ2-related protein interaction network map, and it was found that MYOZ2 may affect the adipogenic differentiation through the interaction of titin-cap (TCAP) and PPARγ. After silencing TCAP, qRT-PCR results showed that compared with the control group, the expression of key adipogenic genes PPARγ and FABP4 were significantly up-regulated (P<0.01); Western Blotting results showed that PPARγ protein was significantly increased (P<0.05); Oil red O staining showed that the number of lipid droplets and the content of triglyceride were significantly increased after TCAP silencing (P<0.05). qRT-PCR was used to detect the expression of TCAP after silencing MYOZ2, and the results showed that the expression of TCAP was significantly increased (P<0.01). In summary, MYOZ2 was highly expressed in longissimus dorsi and lower expressed in subcutaneous fat and liver tissues. In addition, MYOZ2 may regulate the expression of key adipogenic genes PPARγ and FABP4 through the interaction of MYOZ2-TCAP-PPARγ, and to further regulate the expression of fatty acid metabolism related genes SCD, FASN, SREBP1, NR1H3, DGAT1, PNPLA2, HSL, CES1 and CPT1, thus playing an important role in the process of adipogenic differentiation.