Runx2介导TGF-β1调控成釉细胞MMP20基因表达的研究

Runx2 mediates TGF-β1-induced MMP20 expression

目的:研究Runx2在TGF-β1调控小鼠成釉细胞金属基质蛋白酶-20(matrix metalloprotei-nase 20,MMP20)基因表达中的作用。方法:首先利用双荧光素酶基因报告系统分析TGF-β1对MMP20基因启动子转录活性的影响;然后利用染色体免疫共沉淀(Chromatin Immunoprecipitation,ChIP)方法观察Runx2与MMP20特异性结合位点之间的相互作用,并利用基因定点突变和双荧光素酶基因报告系统分析Runx2对MMP20基因启动子转录活性的影响;最后运用小RNA干扰技术使Runx2基因沉默,实时定量RT-PCR技术观察TGF-β1诱导MMP20基因表达的改变。结果:TGF-β1刺激成釉细胞后,MMP20启动子在-87～+23区域转录活性无明显变化外,其他各区域转录活性均增强。利用ChIP研究发现Runx2与MMP20基因核心启动子的特征性序列"TGTGGG"相互作用;将该特征性序列由"TGTGGG"突变为"TGTAAG"后,利用双荧光素酶基因报告系统发现Runx2对MMP20基因启动子转录活性的影响减弱;利用小RNA干扰技术使Runx2基因沉默后,TGF-β1上调MMP20基因表达的作用减弱。结论:TGF-β1通过转录因子Runx2调控成釉细胞MMP20的表达。

AIM： To investigate the regulatory effects of Runx2 on TGF-β1 induced matrix metalloproteinase 20（MMP20） expression in ameloblasts.METHODS： Dual luciferase analysis was used to observe the effects of TGF-β1 on the transcriptional activity of MMP20 promoter,then Chromatin Immunoprecipitation（ChIP） was used to identify the Runx2 protein binding regions in the MMP20 promoter.Furthevmore the Runx2 binding sites were mutated,and Dual luciferase analysis was used to observe the effects of Runx2 on the transcriptional activity of the mutant MMP20 promoter.Finally ameloblasts were transfected with Runx2 siRNA to knockdown Runx2 expression,and the TGF-β1 induced MMP20 expression was detected by real time RT-PCR.RESULTS： Luciferase analysis showed that TGF-β1 enhanced MMP20 promoter activity except the-87-＋23 region.Mutation of the Runx2 binding sites inhibited Runx2-induced MMP20 expression.Knockdown of the transcription factor Runx2 by siRNA inhibited the TGF-β1-induced MMP20 expression.CONCLUTION： TGF-β1 regulates MMP20 gene expression via transcription factor Runx2 in ameloblasts.