Myocardin与ERα协同诱导血管平滑肌细胞分化的机制研究
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摘要
自Olson等人于2001年发现Myocardin以来,其诱导心肌肥厚以及抑制血管平滑肌细胞增殖的机制研究有了很大进展。本文围绕平滑肌细胞(vascular smooth muscle cells, VSMCs)为研究对象,研究转录因子Myocardin与Estrogen receptor a (ERa)在血管平滑肌细胞表型转化中的作用机制。在COS-7细胞系中通过荧光素酶活性实验(luciferase)方法来研究二者在功能上的协同作用；在原代培养的大鼠VSMCs中同样进行功能实验,并对平滑肌细胞分化标志基因在RNA水平以及蛋白水平上进行检测,从而确定细胞分化情况。利用构建的缺失转录激活域(TAD domain)的截短Myocardin真核表达质粒dominant-negative Myocardin (MYCD-dn)与ERa共转染COS-7细胞系,初步探讨Myocardin和ERa在平滑肌细胞分化中的分子作用途径。
Myocardin作为Serum Response Factor (SRF)依赖性反式激活多种平滑肌细胞特异性的转录调节元件,在平滑肌细胞分化中起着重要作用。其主要通过结合SRF,再由SRF结合下游调控目的基因启动子中的CArG box(CC(A/T)6GG)特异性序列,从而激活下游基因的表达。应用生物信息学方法,定位小鼠SM22基因启动子并确定Myocardin在SM22启动子区域-447至+89含有两个CArG Box结合位点。通过PCR方法克隆并构建Myocardin、ERα真核表达质粒以及SM22启动子荧光素酶报告基因质粒。
从SD大鼠中提取主动脉平滑肌细胞,通过RT-PCR方法检测所要研究的内源性目的基因,包括Myocardin、ERα、SM22、α-actin和MHC。将Myocardin、ERa以及SM22-luc质粒转染于体外培养的COS-7和VSMCs中,检测报告基因荧光素酶活性,结果显示Myocardin和ERa能够在功能实验中协同促进SM22-luc的表达。同时检测转染Myocardin和ERa的平滑肌细胞分化标志基因在RNA水平和蛋白质水平上的表达变化,结果显示Myocardin和ERa共转染组细胞的分化标志基因表达有所增强。
为进一步确定Myocardin与ERa在SM22基因启动子上的协同作用机制,构建TAD domain缺失的截短MYCD-dn真核表达质粒,并与ERa共转染于COS-7中。结果显示：MYCD-dn和ERa转染组中,SM22-luc丧失了荧光素酶活性。上述研究结果阐明：ERα对于Myocardin诱导的平滑肌细胞分化有协同促进作用。该促进作用可能是通过Myocardin-CArG box途径来实现的。
Myocardin was firstly discovered by Olson, the mechanism of Myocardin inducing cardiac hypertrophy and inhibiting vascular smooth muscle cell (VSMCs) proliferation has been great progressed. This research focused on the vascular smooth muscle cells (VSMCs) to study the role of transcription factor Myocardin and Estrogen Receptor a (ERa) in regulating phenotypes alteration in the vascular smooth muscle cells. Study the function in COS-7 and VSMCs luciferase experiment, and then the differentiation marker genes were detected at the level of RNA and protein in the VSMCs. The plasmid of dominant-negative Myocardin (MYCD-dn) which lacks the TAD domain was constructed,and then transfected in the COS-7,the mechanism of Myocardin and ERa regulating VSMCs differentiation was initially studied.
Myocardin is a remarkably potent transcriptional coactivator that binds directly to SRF and activates transcription of a subset of SRF-regulated genes encoding contractile proteins. Myocardin binds SRF, and then SRF binds the CArG box (CC(A/T)6GG) of downstream target gene to activate its expression.
According to bioinformatics results, the SM22 luciferase reporter (SM22-luc) gene were constructed successfully which contained promoter region-447 to+89 where two binding sites of Myocardin were determined. The expression plasmids of Myocardin、ERa and SM22 promoter were cloned and constructed by the method of PCR.
The VSMCs were got from SD rat. Target genes were detected by RT-PCR, including Myocardin、ERα、SM22、α-actin and MHC. COS-7 and VSMCs were transfected with the plasmids of Myocardin、ERa and SM22-luc, the luciferase activation was detected. The results were shown:Myocardin and ERa could be co-activator to increase SM22-luc expression. VSMCs were transfected with the plasmids of Myocardin and ERa, the differentiation markers were detected at the level of RNA and protein. The results were shown:Myocardin and ERa also could increase differentiation marker genes expression.
To further investigated the mechanism of how Myocardin and ERa active the SM22 gene promoter, we constructed the plasmid of dominant-negative Myocardin (MYCD-dn) which lacks the TAD domain, and then transfected into COS-7, the luciferase activation was detected. The results were shown:SM22 luciferase activation was lost by cotransfection with MYCD-dn and ERa. The results of the study show:ERαis a co-activator for the major VSMC transcription factor Myocardin, which is required for VSMC differentiation.
Myocardin作为Serum Response Factor (SRF)依赖性反式激活多种平滑肌细胞特异性的转录调节元件,在平滑肌细胞分化中起着重要作用。其主要通过结合SRF,再由SRF结合下游调控目的基因启动子中的CArG box(CC(A/T)6GG)特异性序列,从而激活下游基因的表达。应用生物信息学方法,定位小鼠SM22基因启动子并确定Myocardin在SM22启动子区域-447至+89含有两个CArG Box结合位点。通过PCR方法克隆并构建Myocardin、ERα真核表达质粒以及SM22启动子荧光素酶报告基因质粒。
从SD大鼠中提取主动脉平滑肌细胞,通过RT-PCR方法检测所要研究的内源性目的基因,包括Myocardin、ERα、SM22、α-actin和MHC。将Myocardin、ERa以及SM22-luc质粒转染于体外培养的COS-7和VSMCs中,检测报告基因荧光素酶活性,结果显示Myocardin和ERa能够在功能实验中协同促进SM22-luc的表达。同时检测转染Myocardin和ERa的平滑肌细胞分化标志基因在RNA水平和蛋白质水平上的表达变化,结果显示Myocardin和ERa共转染组细胞的分化标志基因表达有所增强。
为进一步确定Myocardin与ERa在SM22基因启动子上的协同作用机制,构建TAD domain缺失的截短MYCD-dn真核表达质粒,并与ERa共转染于COS-7中。结果显示：MYCD-dn和ERa转染组中,SM22-luc丧失了荧光素酶活性。上述研究结果阐明：ERα对于Myocardin诱导的平滑肌细胞分化有协同促进作用。该促进作用可能是通过Myocardin-CArG box途径来实现的。
Myocardin was firstly discovered by Olson, the mechanism of Myocardin inducing cardiac hypertrophy and inhibiting vascular smooth muscle cell (VSMCs) proliferation has been great progressed. This research focused on the vascular smooth muscle cells (VSMCs) to study the role of transcription factor Myocardin and Estrogen Receptor a (ERa) in regulating phenotypes alteration in the vascular smooth muscle cells. Study the function in COS-7 and VSMCs luciferase experiment, and then the differentiation marker genes were detected at the level of RNA and protein in the VSMCs. The plasmid of dominant-negative Myocardin (MYCD-dn) which lacks the TAD domain was constructed,and then transfected in the COS-7,the mechanism of Myocardin and ERa regulating VSMCs differentiation was initially studied.
Myocardin is a remarkably potent transcriptional coactivator that binds directly to SRF and activates transcription of a subset of SRF-regulated genes encoding contractile proteins. Myocardin binds SRF, and then SRF binds the CArG box (CC(A/T)6GG) of downstream target gene to activate its expression.
According to bioinformatics results, the SM22 luciferase reporter (SM22-luc) gene were constructed successfully which contained promoter region-447 to+89 where two binding sites of Myocardin were determined. The expression plasmids of Myocardin、ERa and SM22 promoter were cloned and constructed by the method of PCR.
The VSMCs were got from SD rat. Target genes were detected by RT-PCR, including Myocardin、ERα、SM22、α-actin and MHC. COS-7 and VSMCs were transfected with the plasmids of Myocardin、ERa and SM22-luc, the luciferase activation was detected. The results were shown:Myocardin and ERa could be co-activator to increase SM22-luc expression. VSMCs were transfected with the plasmids of Myocardin and ERa, the differentiation markers were detected at the level of RNA and protein. The results were shown:Myocardin and ERa also could increase differentiation marker genes expression.
To further investigated the mechanism of how Myocardin and ERa active the SM22 gene promoter, we constructed the plasmid of dominant-negative Myocardin (MYCD-dn) which lacks the TAD domain, and then transfected into COS-7, the luciferase activation was detected. The results were shown:SM22 luciferase activation was lost by cotransfection with MYCD-dn and ERa. The results of the study show:ERαis a co-activator for the major VSMC transcription factor Myocardin, which is required for VSMC differentiation.
引文
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