293T细胞中SQSTM1/p62-GFP的表达及其与LC3在诱导自噬状态下的定位分布

Expression of SQSTM1/p62-GFP and Its Localization and Distribution with LC3 In Autophagy-Induced 293T Cells

为探究SQSTM1/p62蛋白在细胞发生自噬时的定位,以人肺腺癌A549细胞的cDNA为模板,PCR扩增SQSTM1基因(编码p62蛋白)并将其插入pEGFP-N1真核表达质粒。将重组质粒转染进入人胚肾293T细胞中表达p62绿色荧光融合蛋白(GFP-p62),利用Earle’s盐平衡溶液饥饿诱导细胞自噬,Western blotting和激光共聚焦显微镜检测GFP-p62的表达及其与自噬相关蛋白LC3在自噬细胞中的定位。结果发现,重组载体pEGFP-N1-p62转染293T细胞后,293T细胞能高效表达GFP-p62融合蛋白,内源性p62所占细胞内总p62比例很低。饥饿诱导后细胞自噬体形成,LC3-Ⅱ/LC3-Ⅰ的比例明显增高,p62蛋白表达下调。同时还发现,GFP-p62在自噬细胞中仅定位于大多数自噬体且与LC3共定位,小部分自噬体及自噬体以外区域没有明显GFP-p62分布。本研究建立了一种通过p62-GFP与LC3的共定位检测人胚肾293T细胞自噬时p62蛋白和LC3聚集体的有效方法,提示了使用内源性p62低表达细胞作为研究p62和细胞自噬的优势,也将有助于开展关于p62在细胞中定位与其生物学功能关系的研究工作。

To investigate the localization of SQSTM1/p62 protein during autophagy, SQSTM1 gene(p62-coding gene) derived from cDNA of human lung adenocarcinoma A549 cells was amplified and inserted into pEGFP-N1 eukaryotic expression plasmid. The constructed recombinant plasmid was transfected into 293T cells to express p62 green fluorescent fusion protein(GFP-p62). We detected the expression of GFP-p62 by Western blotting and observed its localization with autophagy-related protein LC3 in by laser scanning confocal microscope after inducing cell autophagy by Earle’s salt balance solution. The results showed that GFP-p62 fusion protein was effectively expressed in the transfected 293T cells and the proportion of endogenous p62 protein was very low in total cell p62 protein.After autophagy induced the cell autophagosomes were formed, the ratio of LC3-Ⅱ/LC3-Ⅰ significantly increased and the expression of p62 protein was down-regulated, at the same time we found that GFP-p62 is only localized to most autophagosomes and co-localized with LC3 in autophagy cells, and there is no obvious distribution of GFP-p62 in a small part of autophagosomes and areas outside the autophagosome. This experiment established an effective method to study the co-localization of p62-GFP and LC3 and their aggregates during autophagy in human embryonic kidney 293T cells, prompted the advantage of using low endogenous p62 expression cells to study p62 and cell autophagy, and it was also helpful to study the relevance of p62’s cell location and its biological function.