siRNA沉默ERp57对细胞中CRT表达与定位的影响

Effect of Silencing ERp57 by siRNA on CRT Expression and Localization in A549 Human Lung Cancer Line

探讨ERp57基因表达沉默对人小细胞肺癌A549细胞中CRT表达和定位的影响。利用siRNA技术获得ERp57基因表达沉默的人A549肺癌细胞株,分析该细胞株中ERp57基因以及CRT基因的蛋白表达水平,免疫荧光法检测细胞中CRT的表达和亚细胞定位,荧光法检测细胞凋亡。成功获得ERp57基因表达沉默的人A549肺癌细胞株。在该细胞中,CRT表达上调但仍定位于内质网中。用米托蒽醌处理对照细胞14 h后,可使CRT大量转移到细胞膜表面并发生簇集,但在ERp57表达沉默的细胞中,CRT的膜转移和簇集现象不明显。细胞凋亡分析显示,米托蒽醌处理细胞48 h后,所有细胞均出现凋亡细胞典型细胞核固缩、分裂现象。试验证明抑制ERp57蛋白表达会增加A549肺癌细胞中CRT的含量,但同时也阻断蒽环类药物诱导的CRT膜转移,提示ERp57也是介导肿瘤细胞免疫原性凋亡的重要因子。

It was to investigate the effect of silencing ERp57 on calreticulin（CRT） expression and localization in A549 cells.siRNA technique was used to silence expression of ERp57 gene in human A549 lung cancer line.Western blotting was performed to assay the expression level of ERp57 and CRT genes.The localization of CRT in A549 was tested by immunofluorescrence.Cell apoptosis were determined with fluoresce microscope.An A549 cell line with silenced ERp57 expression was obtained successfully.In this cell line,an increased expression level of CRT was determined,but no change of CRT localization was observed.Immunofluorescence assay indicated that mitoxantrone treatment resulted in CRT exposure on the cell surface quickly in the control cells,but no obvious effect was observed in siRNA-silenced cells.Knocking down ERp57 could facilitate the expression of CRT,but also block the anthracycline-induced CRT membrane translocation,suggesting that ERp57 is also an important factor in mediating immunogenic apoptosis of tumor cells.