Hlcs干扰对C2C12细胞成肌成脂分化后糖酵解基因表达的影响

doi:10.11843/j.issn.0366-6964.2022.10.012

摘要/Abstract

摘要： 旨在研究全羧化酶合成酶（holocarboxylase synthetase，Hlcs）对糖酵解基因表达的影响，为进一步研究细胞糖酵解奠定基础。本试验以C2C12细胞为试验材料，采用siRNA技术干扰Hlcs基因，通过荧光定量PCR和Western blotting检测Hlcs基因干扰对糖酵解基因表达的影响。结果表明，在C2C12细胞分化过程中，Hlcs基因与Pfkm、Pkm、Hk-2基因的表达趋势一致，提示Hlcs基因与糖酵解具有相关性。干扰Hlcs后，细胞分化形成的肌管数量明显少于对照组，糖酵解限速酶Pfkm的mRNA水平显著降低（P<0.05），而显著提高了Pkm的mRNA水平和蛋白表达水平（P<0.05）；生物素作为羧化酶的辅酶，能够显著上调Pkm、Hk-2的mRNA水平（P<0.05）。干扰Hlcs后，细胞分化形成的脂滴数量明显少于对照组，Pfkm的mRNA水平显著升高（P<0.05），且Pkm的mRNA水平和蛋白表达水平显著升高（P<0.05）；生物素的添加能够显著上调Pfkm、Pkm的mRNA水平（P<0.05）。综上所述，在C2C12细胞分化过程中，Hlcs基因与糖酵解基因表达趋势一致，且干扰Hlcs基因可以抑制成肌分化C2C12细胞中Pfkm和Hk-2基因的表达，促进Pkm的表达；而促进成脂分化C2C12细胞中Pfkm和Pkm的表达，抑制Hk-2基因的表达。

关键词: Hlcs基因, siRNA干扰, C2C12细胞, 成肌成脂诱导, 糖酵解基因

Abstract: The aim of this study was to investigate the effect of holocarboxylase synthetase (Hlcs) on glycolytic gene expression and to lay the foundation for further studies on cellular glycolysis. In this experiment, C2C12 cells were used as the experimental material, and siRNA technology was used to interfere with the Hlcs gene, and the effect of Hlcs gene interference on the glycolysis gene expression was detected by fluorescence quantitative PCR and Western blotting. The results showed that, during the differentiation of C2C12 cells, the expression trends of Hlcs genes was consistent with Pfkm, Pkm, Hk-2 genes, suggesting that Hlcs genes were correlated with glycolysis. After interfering with Hlcs, the number of myotubes formed during cell differentiation was significantly less than that in the control group, the mRNA level of the glycolytic rate-limiting enzyme Pfkm was significantly decreased (P<0.05), and the mRNA and protein levels of the Pkm were significantly increased (P<0.05). As a coenzyme of carboxylase, biotin could significantly up-regulate the mRNA levels of key enzymes Pkm and Hk-2 (P<0.05). After interfering with Hlcs, the number of lipid droplets formed by cell differentiation was obviously less than that in the control group, the mRNA level of Pfkm was significantly increased (P<0.05), and the mRNA level and protein expression level of the Pkm were significantly increased (P<0.05); The addition of biotin could significantly up-regulate the mRNA level of Pfkm and Pkm (P<0.05). In summary, during the differentiation of C2C12 cells, Hlcs genes were expressed in the same trend as glycolytic genes, and interfering with Hlcs genes could inhibit the expression of Pfkm and Hk-2 genes and promote the expression of Pkm in myogenic differentiated C2C12 cells; while promoting the expression of Pfkm and Pkm and inhibiting the expression of Hk-2 genes in lipogenic differentiated C2C12 cells.

Key words: Hlcs gene, siRNA interference, C2C12 cells, myogenic and adipogenic induction, glycolysis genes

中图分类号:

S813

代宇星, 史银银, 温作晨, 罗云燕, 祝雪丽, 郑春婷, 李淑英, 洪亮, 张建斌, 郭亮, 蒲蕾. Hlcs干扰对C2C12细胞成肌成脂分化后糖酵解基因表达的影响[J]. 畜牧兽医学报, 2022, 53(10): 3391-3402.

DAI Yuxing,SHI Yinyin,WEN Zuochen,LUO Yunyan,ZHU Xueli,ZHENG Chunting,LI Shuying,HONG Liang,ZHANG Jianbin,GUO Liang,PU Lei. The Effect of Hlcs Interference on Glycolytic Gene Expression in C2C12 Cells after Myogenic Lipogenic Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3391-3402.

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