Functional Analysis of Promoters from Three Subtypes of the PI3K Family and Their Roles in the Regulation of Lipid Metabolism by Insulin in Yellow Catfish Pelteobagrus fulvidraco.
Functional Analysis of Promoters from Three Subtypes of the PI3K Family and Their Roles in the Regulation of Lipid Metabolism by Insulin in Yellow Catfish Pelteobagrus fulvidraco.
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DOI：
10.3390/ijms19010265
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发表时间：
2018-01-16
影响因子：
5.6
通讯作者：
Wu K
中科院分区：
文献类型：
--
作者：
Zhuo MQ;Luo Z;Xu YH;Li DD;Pan YX;Wu K
本研究克隆了黄颡鱼（Pelteobagrus fulvidraco）PI 3 K家族3个亚型（PI 3 KCa、PI 3 KC 2b和PI 3 KC 3）的360、1848和367 bp启动子序列，并对其进行了序列分析。生物信息学分析表明，PI 3 KCa、PI 3 KC 2b和PI 3 KC 3的核心启动子区结构不同。PI 3 KCa和PI 3 KC 2b的启动子区含有CpG岛，但没有CAAT和TATA盒。而PI 3 KC 3基因启动子具有典型的TATA盒和CAAT盒，但没有CpG岛。预测了HNF 1、STAT和NF-κB等转录因子在PI 3 KCa启动子上的结合位点。预测了FOXO 1、PPAR-RXR、STAT、IK 1、HNF 6和HNF 3等转录因子在PI 3 KC 2b启动子上的结合位点，预测了FOXO 1和STAT转录因子在PI 3 KC 3启动子上的结合位点。缺失分析表明，这些转录因子是潜在的调控因子，介导其启动子的活动。随后的突变分析和电泳迁移率变动分析（EMSA）证实HNF 1和IK 1分别与PI 3 KCa和PI 3 KC 2b启动子直接结合，负调控PI 3 KCa和PI 3 KC 2b启动子的活性。相反，FOXO 1直接与PI 3 KC 2b和PI 3 KC 3启动子结合，并正向调节其启动子活性。此外，AS 1842856（AS，一种潜在FOXO 1抑制剂）孵育显著降低了几种PI 3 KC 2b和PI 3 KC 3质粒的相对荧光素酶活性，但未显著影响PI 3 KCa质粒的相对荧光素酶活性。此外，通过使用来自黄颡鱼的原代肝细胞，AS孵育显着下调PI 3 KCa，PI 3 KC 2b和PI 3 KC 3的mRNA水平，并减少三酰甘油（TG）的积累和胰岛素诱导的TG积累，以及参与脂质代谢的几个基因的活性和mRNA水平。因此，本研究提供了新的见解PI 3 Ks的转录调控机制和PI 3 Ks介导的调节脂质代谢的胰岛素在鱼。
In the present study, the length of 360, 1848 and 367 bp sequences of promoters from three subtypes of PI3K family (PI3KCa, PI3KC2b and PI3KC3) of yellow catfish Pelteobagrus fulvidraco were cloned and characterized. Bioinformatics analysis revealed that PI3KCa, PI3KC2b and PI3KC3 had different structures in their core promoter regions. The promoter regions of PI3KCa and PI3KC2b had CpG islands but no CAAT and TATA box. In contrast, the promoter of PI3KC3 had the canonical TATA and CAAT box but no CpG island. The binding sites of several transcription factors, such as HNF1, STAT and NF-κB, were predicted on PI3KCa promoter. The binding sites of transcription factors, such as FOXO1, PPAR-RXR, STAT, IK1, HNF6 and HNF3, were predicted on PI3KC2b promoter and the binding sites of FOXO1 and STAT transcription factors were predicted on PI3KC3 promoter. Deletion analysis indicated that these transcriptional factors were the potential regulators to mediate the activities of their promoters. Subsequent mutation analysis and electrophoretic mobility-shift assay (EMSA) demonstrated that HNF1 and IK1 directly bound with PI3KCa and PI3KC2b promoters and negatively regulated the activities of PI3KCa and PI3KC2b promoters, respectively. Conversely, FOXO1 directly bound with the PI3KC2b and PI3KC3 promoters and positively regulated their promoter activities. In addition, AS1842856 (AS, a potential FOXO1 inhibitor) incubation significantly reduced the relative luciferase activities of several plasmids of PI3KC2b and PI3KC3 but did not significantly influence the relative luciferase activities of the PI3KCa plasmids. Moreover, by using primary hepatocytes from yellow catfish, AS incubation significantly down-regulated the mRNA levels of PI3KCa, PI3KC2b and PI3KC3 and reduced triacylglyceride (TG) accumulation and insulin-induced TG accumulation, as well as the activities and the mRNA levels of several genes involved in lipid metabolism. Thus, the present study offers new insights into the mechanisms for transcriptional regulation of PI3Ks and for PI3Ks-mediated regulation of lipid metabolism by insulin in fish.
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发表时间：
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