葡萄糖调节蛋白78对顺铂治疗舌鳞癌细胞增殖、迁移和侵袭的影响

Effects of GRP78 on the proliferation, migration and invasion of tongue squamous cell carcinoma cells treated with cisplatin

目的探讨葡萄糖调节蛋白78(glucose regulated protein78,GRP78)对舌鳞癌顺铂治疗后生长和转移的影响及机制。方法MTT实验检测舌鳞癌CAL27及舌鳞癌顺铂耐药CAL27DR细胞的增殖情况;应用RNA干扰技术敲减CAL27DR细胞GRP78表达;应用0、2.5、5.0、10.0μmol/L DDP处理CAL27DR及siGRP78细胞;EdU实验检测敲减GRP78表达对细胞增殖能力的影响;Transwell实验检测敲减GRP78表达对细胞侵袭能力的影响;划痕实验检测敲减GRP78表达对细胞迁移能力的影响;免疫荧光实验检测敲减GRP78表达对Cortactin蛋白表达的影响;Western blot实验检测敲减GRP78表达对细胞ERK、p-ERK、AKT、p-AKT、FAK、p-FAK、N-cad和E-cad蛋白表达的影响。结果与CAL27细胞相比,CAL27DR细胞增殖能力显著升高;与对照组细胞相比,siGRP78细胞增殖能力及侵袭转移能力显著降低(P<0.05),Cortactin蛋白表达水平降低,ERK、AKT和FAK磷酸化水平显著降低(P<0.05),N-cad蛋白表达水平降低(P<0.05),E-cad蛋白表达水平升高(P<0.05)。结论GRP78通过ERK、AKT信号通路上调细胞的增殖能力,导致舌鳞癌细胞对DDP的敏感性降低;GRP78介导舌鳞癌细胞EMT的发生并通过活化FAK激酶促进细胞侵袭转移能力升高。

Objective To investigate the effect and mechanism of glucose regulated protein78(GRP78) on the proliferation and metastasis of tongue squamous cell carcinoma(TSCC) treated with cisplatin. Methods MTT assay was used to detect the proliferation of CAL27 and CAL27DR cell. RNA interference was used to knockdown GRP78 expression in CAL27DR cells and then cells were treated with DDP(0 μmol/L, 2.5 μmol/L, 5.0 μmol/L and 10.0 μmol/L)and the proliferation ability was detected by EdU. Transwell assay was used to detect the invasive potential and scratch assay was used to detect the migration potential in CAL27DR and siGRP78 cells. Immunofluorescence assay was used to detect the expression of Cortactin. Western blot assay was used to detect the protein expression of ERK, p-ERK, AKT, p-AKT, FAK, p-FAK, N-cad and E-cad in CAL27DR and siGRP78 cells. Results MTT result showed that the proliferation potential was increased obviously in CAL27DR cells compared with CAL27 cells. EdU assay result showed that the proliferation potential was decreased obviously in siGRP78 cells compared with vector(P<0.05).Transwell and scratch assay results showed that the invasive and migration potential was also decreased in siGRP78 cells compared with vector(P<0.05). Immunofluorescence assay result showed that Cortactin protein expression was decreased in siGRP78 cells compared with vector. The phosphorylation levels of ERK, AKT and FAK were significantly increased in siGRP78 cells compared with vector(P<0.05). The protein expression of N-cad was increased in siGRP78 cells compared with vector and the protein expression of N-cad has the opposite trend(P<0.05). Conclusion GRP78 up-regulates cell proliferation through ERK and AKT kinases, resulting in decreased sensitivity of cells to DDP, and mediates the occurrence of EMT and promotes cell invasion and metastasis by activating FAK kinase in tongue squamous cells.