三乙烯四胺对c-myc启动区转录活性的调节作用

Regulative effect of triethylene tetramine on transcription of c-myc promoter

目的探讨三乙烯四胺(TETA)如何调节c-myc基因表达。方法利用圆二色谱分析TETA对c-myc启动区核酸酶超敏元件Ⅲ1(c-mycNHEⅢ1)碱基序列形成的G-四链体DNA(G4-DNA)结构稳定性的影响。同时,分别构建含c-mycNHEⅢ1的野生型和突变型的c-myc启动区荧光报告质粒,并分别转染HEK293细胞24h后,再接种到96孔板,TETA以终浓度0,0.1,1.0,10及100μmol·L-1处理8h,测定荧光素酶活性,计算TETA对其转录活性抑制率。结果圆二色谱实验结果表明,TETA5μmol·L-1就能够进一步增强c-myc NHEⅢ1在240nm处的负峰和260nm处的正峰的形成,即增强G4-DNA结构的稳定性。报告基因分析结果表明,TETA能够浓度依赖性地抑制野生型c-myc启动区荧光素酶基因的表达,但对突变型c-myc启动区荧光素酶基因的抑制能力明显下降。在TETA1nmol·L-1作用时,对突变型c-myc启动区荧光素酶基因抑制率仅为4.3%,而对野生型的抑制率还高达30.4%。结论TETA可能通过稳定c-mycNHEⅢ1序列形成的G4-DNA结构调节基因的表达。

AIM To explore effects of triethylene tetramine （TETA） on the transcription of c-myc promoter. METHODS Circular dichroism （CD） was collected to identify the influence of TETA on the stability of G-quadruplex formed by c-myc promoter region nuclease hypersensitive element Ⅲ1 （c-myc NHE Ⅲ1） sequence. Furthermore the wild and mutant reporter gene plasmids containing c-myc NHE Ⅲ1 sequence were constructed,the 2 plasmids were transfected into HEK293 cells respectively for 24 h. The transfected cells were replated into 96 wells plate,and treated with different concentrations of TETA （0,0.1,1,10 and 100 μmol·L^-1） for about 8 h,the luciferase activity was determined with its substrate BrightGlo. The inhibition rate of TETA on the reporter gene was calculated by the luciferase activity. RESULTS TETA 5 μmol·L^-1 will be able to further enhance absorption at 240 nm （the negative peak） and 260 nm （positive peak）. So the TETA maybe assistant the G4-DNA structures formation and increase the stability in the c-myc NHE Ⅲ1 region. Furthermore,from the results of reporter gene analysis,TETA could inhibit the expression of reporter gene in a dose-dependent manner,but for the mutated sequence,the inhibition of TETA on the expression of reporter gene was decreased significantly. With TETA 1 nmol·L^-1 treated,inhibition rate of mutant-type report gene expression was only 4.3%,while the inhibition rate of wild-type was as high as 30.4%. CONCLUSION TETA has negative regulatory effect on c-myc promoter through enhancing the stability of G-quadruplex formed by the sequence of nuclease hypersensitive element Ⅲ1.