Lrtm1基因在成肌分化过程中的功能探索

Functional exploration of Lrtm1 gene in myogenic differentiation

摘要:

目的：观察成肌分化过程中C2C12细胞Lrtm1表达；构建小鼠Lrtm1慢病毒过表达载体；筛选稳定表达外源Lrtm1细胞株诱导其分化后观察对成肌分化标志基因Myogenin、Myosin的影响；发现Lrtm1蛋白质表达水平受调控的机制。方法：诱导C2C12细胞分化用Hoechst染色细胞核；将小鼠Lrtm1基因连接到慢病毒载体质粒pLJM1-GFP上并进行慢病毒包装；筛选稳定过表达Lrtm1基因的C2C12细胞系，诱导分化Lrtm1-DDK组、GFP组检测对成肌分化标志基因Myogenin、Myosin表达的影响；MG132处理C2C12-Lrtm1-DDK细胞系后，观察内源性Lrtm1蛋白质表达。结果：诱导分化过程中，肌管形成，Lrmt1、Myosin mRNA水平随分化的进行表达上升；成功构建重组pLJM1-Lrtm1-DDK表达载体；成功筛选稳定表达外源Lrtm1细胞株；诱导稳定细胞系分化后，real-time PCR在144 h时显示Lrtm1-DDK组（272.1±18.22）Myogenin mRNA水平较GFP组（332.41±41.21）减少（P＜0.05）；Lrtm1-DDK组（76.11±10.47）Myogenin mRNA水平较GFP组（145.51±10.02）减少（P＜0.05）；C2C12细胞中Lrtm1蛋白质被蛋白酶体降解。结论：Lrtm1基因在成肌分化过程中高表达；过表达Lrtm1基因部分抑制了Myogenin、Myosin基因的表达；Lrtm1蛋白质表达水平受蛋白酶体降解调控。

Abstract:

Objective：To observe Lrtm1 expression in C2C12 cells，to construct a lentiviral vector overexpressing Lrtm1 gene effectively in C2C12 cells，to determine its role in myoblast differentiation and to explore the mechanism by which the expression level of Lrtm1 protein is regulated. Methods：The differentiation of C2C12 cells was induced and the nucleus was stained by Hoechst. The mouse Lrtm1 gene was recombined with lentiviral vectors pLJM1-GFP. C2C12 cells which overexpress ectopic Lrtm1 were selected. After treating with C2C12-Lrtm1-DDK cell lines with MG132，the expression of endogenous Lrtm1 protein was observed. Results：Myotubes had formed during its differentiation. The expression of Lrmt1 and Myosin mRNA increased during differentiation.The recombinant pLJM1-Lrtm1-DDK vector was successfully constructed and stable cell lines were screened. The results of real-time PCR at 144 h showed that the Myogenin mRNA level in the Lrtm1-DDK group（272.1±18.22） was lower than that in the GFP group（332.41±41.21）（P<0.05）. The Myosin mRNA level was significantly lower in Lrtm1-DDK group（76.11±10.47） than in GFP group（145.51±10.02） （P＜0.05）. Lrtm1 protein was degraded by the proteasome in C2C12 cells. Conclusion：Lrtm1 gene is highly expressed during myogenic differentiation. Overexpression of Lrtm1 partially inhibits the expression of Myogenin and Myosin. Lrtm1 protein is degraded by the proteasome.