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目的探讨瘦素抑制小鼠过氧化物酶体增殖物激活受体(PPAR)γ1基因表达的可能机制。方法从UCSC数据库获得小鼠PPARγ1基因转录起始点上游约3kbp的序列,分别运用Patch和Signal Scan在线软件分析该序列,筛选出两组结果中相同的转录因子及相应的结合位点,并找出其中与脂质代谢和脂肪细胞分化相关的转录因子,采用Western blot和瞬时转染实验验证。结果经过筛选,GATA结合蛋白2(GATA-2)是与脂质代谢和脂肪细胞分化相关的转录因子。Western blot结果显示,经瘦素处理的肝星状细胞的GATA-2表达升高(P<0.05);在转染pPPARγ1(-2333)Luc的肝星状细胞中,瘦素处理后的荧光素酶活性降低(P<0.05),而在转染pPPARγ1(-1823)Luc、pPPARγ1(-1313)Luc的肝星状细胞中,瘦素处理后的荧光素酶活性无明显变化(P>0.05)。与转染pPPARγ1(-2333)Luc的肝星状细胞相比,转染pPPARγ1(GATA mut)Luc的肝星状细胞荧光素酶活性升高(P<0.05)。结论 GATA-2通过与PPARγ1的5′端转录起始点上游-1823~-2333的区域结合参与瘦素对小鼠PPARγ1基因表达的抑制。
Objective To investigate the possible mechanism of leptin in inhibiting the expression of peroxisome proliferator - activated receptor (PPAR) γ1 in mice. Methods The sequence of about 3kbp upstream of PPARγ1 gene transcription was obtained from UCSC database. The sequence was analyzed by Patch and Signal Scan online software, and the same transcription factor and its corresponding binding sites were screened out in two groups Among them, the transcription factors related to lipid metabolism and adipocyte differentiation were verified by Western blot and transient transfection experiments. Results After screening, GATA-2 (GATA-2) was a transcription factor involved in lipid metabolism and adipocyte differentiation. Western blot results showed that the expression of GATA-2 in leptin-treated hepatic stellate cells was increased (P <0.05). In the hepatic stellate cells transfected with pPPARγ1 (-2333) Luc, leptin-treated fluorescein (P <0.05). However, there was no significant change in the luciferase activity of HSCs transfected with pPPARγ1 (-1823) Luc and pPPARγ1 (-1313) Luc (P> 0.05) . Compared with hepatic stellate cells transfected with pPPARγ1 (-2333) Luc, hepatic stellate cells transfected with pPPARγ1 (GATA mut) Luc had higher luciferase activity (P <0.05). Conclusion GATA-2 participates in the inhibitory effect of leptin on the PPARγ1 gene expression in mice by binding to the -1823 ~ -2333 region upstream of the 5 ’transcription start site of PPARγ1.