Identification of direct forkhead box O1 targets involved in palmitate-induced apoptosis in clonal insulin-secreting cells using chromatin immunoprecipitation coupled to DNA selection and ligation
Identification of direct forkhead box O1 targets involved in palmitate-induced apoptosis in clonal insulin-secreting cells using chromatin immunoprecipitation coupled to DNA selection and ligation
复制标题
使用染色质免疫沉淀结合 DNA 选择和连接鉴定参与棕榈酸酯诱导克隆胰岛素分泌细胞凋亡的直接叉头盒 O1 靶标
DOI：
10.1007/s00125-012-2643-9
复制
发表时间：
2012-10-01
期刊：
影响因子：
8.2
通讯作者：
Han, X.
中科院分区：
文献类型：
--
作者：
Lin, H. Y.;Yin, Y.;Han, X.
目的/假设转录因子，叉头盒（FOX）O 1，参与脂肪酸诱导的胰腺β细胞凋亡，但确切的机制知之甚少。本研究旨在确定FOXO 1的直接下游靶点参与棕榈酸诱导的胰腺β细胞株MIN 6的凋亡。方法采用染色质免疫沉淀（ChIP）结合DNA选择和连接技术（ChIP-DSL）鉴定FOXO 1的直接靶点。实时荧光定量PCR检测mRNA水平。ChIP-DSL结果分别用ChIP-PCR和荧光素酶试验进行验证。TUNEL法测定细胞凋亡率，并通过评分细胞与固缩nucleus.ResultsWe确定了189个靶基因，并选择了106个目标在MIN 6细胞与棕榈酸酯处理的表达分析。结果显示，6个基因显著上调，4个基因下调。通过ChIP-PCR测定FOXO 1与启动子的结合，并通过荧光素酶测定证实。在10个上调和下调的基因中，A930038 C 07 Rik在8周龄ddb/db小鼠中的mRNA表达显著降低，Ppa 1的mRNA表达显著升高。细胞凋亡实验表明，过量表达RIKEN cDNA A930038 C 07（A930038 C 07 Rik）可显著增强棕榈酸诱导的细胞凋亡，而焦磷酸酶（无机）1（PPA 1）可部分保护细胞免于凋亡。敲低PPA 1，此外，显着增加apoptosis.Conclusions/interpretationWe确定了第一次FOXO 1的目标在MIN 6细胞棕榈酸酯处理，从而揭示了重要的作用，A930038 C 07里克和PPA 1棕榈酸酯诱导的细胞凋亡。这些结果揭示了棕榈酸诱导胰腺β细胞凋亡的机制。
Aims/hypothesisThe transcription factor, forkhead box (FOX)O1, is involved in fatty acid-induced apoptosis in pancreatic beta cells, but the precise mechanism is poorly understood. We aimed to identify which direct downstream targets of FOXO1 are involved in palmitate-induced apoptosis in the pancreatic beta cell line MIN6.MethodsChromatin immunoprecipitation (ChIP) coupled to a DNA selection and ligation technique (ChIP–DSL) was used to identify the direct targets of FOXO1. The mRNA level was examined by real-time PCR assay. The ChIP–DSL results were verified using ChIP-PCR and luciferase assay, respectively. The cell apoptosis rate was determined by TUNEL assay and by scoring cells with pycnotic nuclei.ResultsWe identified 189 target genes and selected 106 targets for expression analysis in MIN6 cells treated with palmitate. The results showed that six genes were significantly upregulated and four were downregulated. Binding of FOXO1 to the promoters was determined by ChIP-PCR and confirmed by luciferase assay. Among the ten up- and downregulated genes, mRNA expression ofA930038C07Rikwas significantly decreased and that ofPpa1was increased in 8-week-olddb/dbmice. The apoptosis assay showed that overproduction of the protein ‘RIKEN cDNA A930038C07’ (A930038C07Rik) drastically enhanced palmitate-induced apoptosis, while pyrophosphatase (inorganic) 1 (PPA1) partially protected the cells from apoptosis. Knockdown of PPA1, moreover, significantly increased apoptosis.Conclusions/interpretationWe identified for the first time FOXO1 targets in MIN6 cells treated with palmitate, thus revealing the important roles of A930038C07Rik and PPA1 in palmitate-induced cell apoptosis. These results shed light on the mechanisms of palmitate-induced apoptosis in pancreatic beta cells.