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IGF-1对肌肉特异性组蛋白甲基转移酶SMYD1的表达调控研究
A Study about the Regulatory Role of IGF-1 on the Expression of Muscle-specific Histone Methyltransferase SMYD1
【作者】 王娟；
【导师】 刘明耀；
【作者基本信息】 华东师范大学 ， 生物医学， 2010， 硕士
【摘要】 SMYD1是一个在人类心脏和肌肉组织中特异表达的组蛋白甲基转移酶蛋白,在心脏和肌肉发育过程中起关键作用。SMYD1作为组蛋白H3K4的甲基转移酶能激活下游基因的转录,但目前还没有发现上游细胞外因子如何调控SMYD1基因自身的表达。由于IGF-1(胰岛素样生长因子-1)能促进心肌和骨骼肌的发育,加速肌肉损伤修复过程,我们认为IGF-1很有可能通过激活SMYD1的表达促进肌肉分化.通过Western杂交实验,发现IGF-1处理C2C12细胞,SMYD1蛋白表达水平随处理时间逐步升高,同时SRF(血清应答因子)蛋白和Myogenin(成肌素)的表达也呈现类似的趋势；通过构建不同长度的SMYD1基因启动子荧光素酶报告基因载体,发现SMYD1基因启动子上IGF-1的应答区域位于-620--110 bp;EMSA实验表明SRF结合在SMYD1启动子的CArG位点,IGF-1能够促进SRF与Smydl启动子的结合,将启动子上的CArG元件突变,IGF-1对SMYD1启动子的激活效应被削弱；可见IGF-1能够上调SMYD1在C2C12细胞中的转录和翻译水平,而这种调节作用是部分通过SRF与Smyd1启动子上CArG位点结合实现的。此外,通过荧光素酶报告基因分析,发现SMYD1能够激活肌肉标志因子——肌肉肌酸激酶(MCK)基因的启动子活性,而且与MyoD基因存在协同激活的效应,证明SMYD1可能通过与MyoD的协同作用,促进肌肉分化。
【Abstract】 IGF-1, insulin-like growth factor 1, plays an essential role in heart and skeletal muscle development. SMYD1, a heart and muscle-specific expressed gene, has been demonstrated to be a regulator of cardiomyocyte differentiation. However, it is not fully understood that how upstream signaling regulates SMYD1 expression. The research about the relation of IGF-1 and SMYDl will helpful to the study of the function of SMYD1 in muscle development and the study of related diseases. In this article, we try to demonstrate how IGF-1 regulates the expression of SMYD1. With RT-PCR analysis, the mRNA expression level of Smydl gene was upregulated in C2C12 cells during myogenic differentiation. The protein level of SMYD1 was upregulated as well as SRF, when C2C12 cells were treated with IGF-1 at different time point. In order to determine the IGF-1 response elements in SMYD1 promoter, we constructed a serial of different length of SMYD1 gene promoter luciferase reporter vector. By luciferase experiment, we discovered that the response element of IGF-1 in SMYD1 gene promoter were located at-620--110 bp. Next, using EMSA experiment, we found that SRF could bind to CArG site in SMYD1 promoter, while IGF-1 could regulate SRF binding in Smydl promoter. Then, using of luciferase experiments, we showed that the CArG element involved the regulation of IGF-1 on Smydl in transcriptional level partly. These findings suggested that IGF-1 could increase SMYD1 expression in transcription level in C2C12 cells.