小鼠β细胞系MIN6细胞糖脂毒性模型中Siah1表达变化的研究

Expression of Siah1 in the Glucolipotoxicity Model of MIN6 Cells

目的： Siah1作为一种E3泛素连接酶，在调控细胞凋亡过程中发挥着重要作用，文章旨在在细胞系水平构建胰岛β细胞糖脂毒性模型，检测其中Siah1的表达水平，并预测此模型下Siah1表达的调控机制。方法用高糖高脂联合处理小鼠胰岛β细胞系MIN6细胞，葡萄糖刺激的胰岛素分泌（ glucose stimula－ted insulin secretion， GSIS）实验检测MIN6细胞胰岛素分泌功能，酸乙醇抽提并检测MIN6细胞内胰岛素含量， Western印迹实验检测Parp1蛋白的剪切情况及Siah1的蛋白表达水平，定量RT－PCR分析Siah1的mR－NA水平。借助miRanda软件预测小鼠、大鼠、人这3个种属中可能调控Siah1表达的microRNAs （ miR－NAs），并在种属间进行对比分析。结果高糖高脂处理可损伤MIN6细胞的GSIS功能、减少MIN6细胞胰岛素含量，并能导致Parp1蛋白的剪切程度显著增加。该模型下， Siah1的蛋白表达水平增高，而mRNA水平几乎没有变化，这提示此过程中Siah1主要受转录后水平的调控。用miRanda软件预测可能调控Siah1表达的miRNAs，其中有19个miRNAs的结合位点在小鼠、大鼠及人这3个种属中具有同源性。结论糖脂毒性可导致MIN6细胞功能障碍及凋亡，并能增高MIN6细胞中Siah1的蛋白表达水平，而不影响Siah1的mRNA水平。

Objective As an E3-ubiquitin ligase, Siah1 plays an important role in regulating cell apoptosis.This study aimed to construct a cellular model of glucolipotoxicity and to measure the expression level of Siah1 and explore its regulation mechanism in this model.Methods MIN6 cells were incubated with high glucose and palmitate, whose insulin secretion and insulin content were later assessed by glucose-stimulated insulin secretion （ GSIS） assay.Western blot assay was carried out to evaluate the protein levels of cleaved Parp1 and Siah1.Quantitative RT-PCR was performed to analyze the mRNA level of Siah1.The microRNAs （ miRNAs） regulating Siah1 in the mouse, rat and human were predicted and screened by bioinformatics analysis.Results MIN6 cells incubated with high glucose and palmitate displayed reduced GSIS and insulin contents and increased protein level of cleaved Parp1.The protein level of Siah1 was increased by glucolipotoxicity, though the mRNA level of Siah1 changed little, suggesting that the expression of Siah1 was mainly regulated in post-transcriptional level under the condition of glucolipotoxicity.Among the predicted miRNAs that could regulate Siah1, 19 miRNAs had the highly conserved potential binding sites for Siah1 within 3′UTR in the mouse, rat and human.Conclusion In MIN6 cells, glucolipotocity can lead to βcell dysfunction as well as apoptosis and increase the protein level of Siah1 without influence on its mRNA level.