HepaCAM蛋白通过exosomes途径分泌到细胞外并抑制肿瘤细胞增殖

HepaCAM secreted by renal cell carcinoma 786-0 cells through exosomes pathway inhibits cell proliferation

目的探讨肝细胞黏附分子(hepatocyte cell adhesion molecule,hepaCAM)在肾癌786-0细胞中通过exo-somes途径分泌到细胞外及是否可以抑制肿瘤细胞增殖。方法将携带hepaCAM基因的重组腺病毒质粒瞬时转染786-0细胞,Western blot鉴定hepaCAM基因在786-0中的表达;莫能星(monensin)和二甲基阿米洛利(DMA)处理转染后肿瘤细胞,检测肿瘤细胞上清液及exosomes中hepaCAM蛋白的量的变化。MTT法检测hepaCAM蛋白是否可以抑制肿瘤细胞增殖。结果成功构建并转染hepaCAM重组腺病毒质粒,在肿瘤细胞上清液中检测到hepaCAM蛋白的分泌,经药物处理后,莫能星明显增加了转染后肿瘤细胞的hepaCAM蛋白的分泌量,而二甲基阿米洛利处理转染后肿瘤细胞,能显著抑制细胞上清液中hepaCAM的分泌量。Western blot进一步证明hepaCAM蛋白存在于exosomes上。成功转染hepaCAM基因的肾癌细胞分泌的exosomes明显抑制了肿瘤细胞增殖。结论 hepaCAM蛋白可能通过exosomes途径分泌到肿瘤细胞外并抑制肿瘤细胞增殖。

Objective To investigate that whether hepatocyte cell adhesion molecule（hepaCAM） is actively secreted from renal cell carcinoma 786-0 cells by a pathway involving exosomes and whether hepaCAM could inhibit proliferation in 786-0 cells.Methods A recombinant adenovirus vector plasmid containing hepaCAM was transfected into 786-0 cells.The expression of hepaCAM was detected by Western blotting.The transfected cells were treated with monensin and 5-（N-N-Dimetyl）-amiloride hydrochloride（DMA）,and then extracellular culture medium and exosomes were used to investigate the content of hepaCAM by Bradford protein assay.MTT assay was used to detect the cell viability after treatment of exosomes cotaining hepaCAM.Results HepaCAM adenovirus vector plasmid was successfully constructed and stably transfected into 786-0 cells.Monensin enhanced the release of hepaCAM while DMA inhibited the process in the transfected 786-0 cells.Exosomes purityed from the supernatant of transfected 786-0 cells inhibited the proliferation of 786-0 cells obvously.Conclusion HepaCAM is secreted from renal cell carcinoma cells by a pathway involving exosomes,and inhibits cell proliferation at the same time.