摘要目的 探讨固醇调节元件结合蛋白1c(SREBP-1c)抑制骨骼肌细胞胰岛素受体底物1 (IRS-1)基因转录的具体分子机制.方法 将SREBP-1c表达质粒、IRS-1启动子荧光素酶报告质粒及海肾质粒胸腺嘧啶核苷激酶启动子(pRL-TK)采用Lipofectamin 2000共转染L6细胞,以pCDNA3.1空质粒为对照,36 h后裂解细胞按双荧光素酶报告基因检测试剂盒说明书检测荧光素酶.将表达SREBP-1c的腺病毒感染L6肌管细胞,以表达绿色荧光蛋白(GFP)的腺病毒作对照,感染48 h后收取细胞抽提核蛋白,进行凝胶迁移滞后实验(EMSA)和染色质免疫共沉淀(CHIP)实验,分析SREBP-1c转录因子与IRS-1启动子区域的相互作用.组间比较采用双因素方差分析.结果 荧光素酶截断实验显示IRS-1启动子区域-450~-210 bp为SREBP-1c的作用范围.序列分析显示IRS-1启动子-302~-292 bp处存在SREBP-1c潜在的结合序列GCCTCCCGAG,该序列突变为TGTTAAATTA,荧光素酶实验显示SREBP-1c抑制野生型IRS-1启动子活性,而对突变型IRS-1启动子无抑制作用(分别为7.03± 1.28比19.09±2.45、3.55±1.68比3.96±1.09,F=114.437、0.251,均P>0.05);EMSA结果显示SREBP-1c蛋白与野生型探针直接结合,而不被突变探针所竞争.CHIP结果显示SREBP-1c可直接结合到IRS-1的启动子区域,定量分析显示PA组细胞SREBP-1c结合到IRS-1启动子区域的量为对照组的2倍, SREBP-1c过表达组为GFP组的5倍(分别为2.15±0.03比1.07±0.24,5.48±1.28比0.86±0.19,t=6.8775、5.495,均P<0.05).结论 SREBP-1c通过直接结合到IRS-1启动子区域的非典型固醇反应元件序列抑制IRS-1基因转录表达,继而影响胰岛素信号通路的转导.

abstractsObjective To investigate the molecular mechanism of sterol regulatory element binding protein-1c (SREBP-1c) suppressing insulin receptor substrate-1(IRS-1) in skeletal muscle cells. Methods Luciferase plasmid for rat IRS-1 promoter, expression plasmid of SREBP-1c and pRL-TK renilla plasmid were cotransfected into L6 cells using Lipofectamine 2000 (Invitrogen). After transfection for 36 h, luciferase activity was measured using the dual-luciferase reporter assay system following the manufacturer' s instructions. L6 myotubes were infected with adenoviral vectors expressing SREBP-1c. Adenovirus expressing green fluorescent protein (GFP) was used as control. The cells were harvested for nucleus protein after infection for 48 h. Electrophoretic mobility shift assay (EMSA) was performed using a Light Shift Chemiluminescent EMSA Kit. Chromatin immunoprecipitation assay was performed by CHIP Kit. The interaction between the transcription factor SREBP-1c and the promoter region of IRS-1 was assessed by above experiments. Differences among the groups were determined using two-way ANOVA. Results The luciferase deletion studies suggested the region from-450 to-210 bp on the IRS-1 promoter was a potential target region for SREBP-1c. Sequence analysis showed that the target region of the rat IRS-1 promoter gene contained a potential binding site (GCCTCCCGAG), which was located between-302 and-292 bp. PCR site-directed mutagenesis (TGTTAAATTA) was generated and analyzed using a luciferase assay. The transcriptional activity of wild-type IRS-1 promoter was dramatically down-regulated by SREBP-1c, whereas SREBP-1c had no effect on the IRS-1 promoter bearing mutation of SREBP-1c binding site(7.03 ± 1.28 vs 19.09 ± 2.45,3.55 ± 1.68 vs 3.96 ± 1.09,F=114.437,0.251,all P>0.05). The EMSA results showed that the labeled wild-type probe successfully formed a complex with nuclear proteins. But the unlabelled mutant probe did not interfere the complex. The results from CHIP assay showed that SREBP-1c could bind directly to the IRS-1 promoter region. The quantity of SREBP-1c protein binding to the IRS-1 promoter was two times of control under PA conditions in L6 cells or five times of control when SREBP-1c was over-expressed (2.15 ± 0.03 vs 1.07 ± 0.24,5.48 ± 1.28 vs 0.86 ± 0.19,t=6.877,5.495,all P<0.05). Conclusion SREBP-1c could directly bind to the atypical SRE sequence in the promoter region of IRS-1, suppressing IRS-1 expression and the subsequent insulin signaling pathway.