口腔癌细胞通过传递性内质网应激影响胰岛β细胞功能的初探

Oral cancer cells affect pancreatic β-cell function through transmissible endoplasmic reticulum stress

目的探究口腔癌细胞是否通过传递性内质网应激(TERS)影响胰岛β细胞功能。方法选用衣霉素(TM)作为内质网应激(ERS)剂、人口腔鳞癌细胞系CAl-27、SCC-25作为供体细胞、小鼠胰岛素瘤6细胞系MIN6作为受体细胞,采用实时荧光定量聚合酶链反应(qPCR)、蛋白免疫印迹(WB)检测ERS标志物及胰岛素表达情况,通过脱氧核苷酸末端转移酶介导的脱氧尿苷三磷酸缺口末端标记(TUNEL)技术检测细胞凋亡水平,采用克隆形成检测细胞增殖能力,采用酶联免疫吸附测定(ELISA)、二喹啉甲酸(BCA)试剂盒检测胰岛β细胞分泌功能。结果对MIN6细胞施加TM刺激,通过qPCR、WB发现,ERS标志物上调,这意味着MIN6细胞能产生ERS。将ERS的口腔癌细胞的上清液加入MIN6细胞,通过qPCR、WB发现ERS的口腔癌细胞可诱导MIN6细胞出现ERS,即发生TERS现象;通过TUNEL实验,发现TERS的MIN6细胞凋亡增加;通过克隆形成实验,发现TERS的MIN6细胞增殖能力下降;通过qPCR、WB发现,TERS的MIN6细胞的胰岛素合成减少,在翻译水平抑制胰岛素的合成;通过ELISA、BCA发现,TERS的MIN6细胞分泌功能下降。结论口腔癌细胞可通过TERS引起胰岛β细胞应激,导致其凋亡增加,存活能力下降,合成及分泌胰岛素能力下降。

Objective In this study,we aimed to investigate whether oral cancer cells affect pancreaticβ-cells function through transmissible endoplasmic reticulum stress(TERS).Methods Tunicamycin(TM)was selected as the endoplasmic reticulum stress(ERS)inducer.The human oral cancer cell lines CAl-27 and SCC-25 were selected as the donor cells,and mouse insulinoma 6(MIN6)cell lines were chosen as the recipient cells.Quantitative real-time polymerase chain reaction(qPCR)and Western blot(WB)analysis were used to detect ERS markers and insulin expression.The TdT-mediated dUTP nick-end labeling(TUNEL)method was applied to detect apoptosis levels.The clone formation method was utilized to detect cell proliferation capability.The secretory function of pancreaticβ-cells was detected with an enzyme linked immunosorbent assay(ELISA)kit and a bicinchoninic acid(BCA)kit.Results The MIN6 cells were subjected to TM stimulation.qPCR and WB analysis revealed that ERS markers were upregulated.This result implied that the MIN6 cells can induce ERS.The supernatant of oral cancer cells under ERS was added to the MIN6 cells.qPCR and WB analysis showed that the oral cancer cells that had been subjected to ERS could induce ERS in the MIN6 cells,that is,the phenomenon of TERS occurred.The TUNEL assay indicated that the apoptosis of the MIN6 cells increased under TERS.The clone formation assay demonstrated that the proliferation capability of the MIN6 cells decreased under TERS.qPCR and WB analysis revealed that under TERS,insulin synthesis by the MIN6 cells decreased and insulin synthesis was inhibited at the translation level.The ELISA and BCA kits demonstrated that insulin secretion by the MIN6 cells was reduced under TERS.Conclusion Oral cancer cells can affect pancreaticβ-cells through TERS,resulting in increased apoptosis,decreased viability,and reduced insulin secretion and synthesis capability.