低氧诱导胰腺癌细胞通过外泌体途径分泌高迁移率族蛋白1

Hypoxia induces HMGB1 secretion through exosomes in pancreatic cancer cells

目的:研究低氧诱导条件下胰腺癌细胞中高迁移率族蛋白1(high mobility group box-1 protein,HMGB1)的分泌途径。方法:经GEO和TCGA数据库分析HMGB1在胰腺癌样本中的表达;低氧处理胰腺癌PaTu8988细胞0,6,12,24,48 h,免疫印迹法检测培养上清液中HMGB1的含量;采用外泌体提取试剂盒分离常氧和低氧培养液中的外泌体,运用透射电镜观察外泌体膜结构,运用免疫印迹法检测外泌体中的HMGB1以及外泌体标志蛋白CD9、CD63、CD81以及HSP70的表达;利用免疫荧光法和激光扫描共聚焦显微镜观察HMGB1在PaTu8988细胞中的定位情况。结果:数据库分析结果表明,与健康胰腺组织相比,HMGB1在胰腺癌组织中呈高表达(P<0.01),且高表达者生存期明显短于低表达者(P<0.05)。低氧处理48 h,细胞培养上清液中的HMGB1含量最多,明显高于6,12,24 h。透射电镜观察到培养上清液中有微囊泡结构,直径在100 nm左右,蛋白质免疫印迹法检测到外泌体膜标志蛋白CD9,CD63,CD81,HSP70的表达和外泌体中HMGB1的表达。免疫荧光结果显示,低氧情况下HMGB1和外泌体共定位增多。结论:低氧处理条件下,胰腺癌细胞中HMGB1可通过外泌体途径释放到细胞外。

Objective: To study the secretory pathway of high mobility group box-1 protein( HMGB1) in hypoxia-induced human pancreatic cancer cells. Methods: The expression of HMGB1 in pancreatic cancer was analyzed by GEO and TCGA database. Western blotting was used to detect the content of HMGB1 in the supernatant of pancreatic cancer PaTu8988 cells under different hypoxic time points( 0,6, 12,24,48 h);the exosomes in pancreatic cancer PaTu8988 cells culture medium under normal and hypoxic conditions were separated by exosome extraction kit. The exosomes were identified by transmission electron microscopy and Western blotting,and HMGB1 in exosomes was detected by Western blotting. Immunofluorescence staining was used to observe the distribution of HMGB1 in cells by confocal microscope under normoxia and hypoxia. Results: The results of GEO and TCGA database showed that HMGB1 was highly expressed in pancreatic cancer tissues compared with normal pancreatic tissue ( P < 0. 01), and the survival time of high-expression patients was significantly shorter than that of low-expression patients( P < 0. 05). After hypoxic treatment for 48 h,the expression of HMGB1 in the cell culture supernatant was the highest,significantly higher than that of 6,12,24 h. Transmission electron microscopy showed that the culture had microvesicle structure with a diameter of about 100 nm. Western blotting results showed the expression of exosome membrane markers CD9,CD63,CD81,HSP70 and the expression of HMGB1 in exosomes. Immunofluorescence results showed that HMGB1 and exosomes were co-localized in hypoxia. Conclusion: HMGB1 could be released through the exosomal pathway in PaTu8988 pancreatic cancer cells under hypoxia.