14-3-3ζ蛋白诱导NK/T细胞淋巴瘤吉西他滨耐药的机制研究

14-3-3ζprotein mediates gemcitabine resistance in NK/T-cell lymphoma

目的探讨14-3-3ζ蛋白在结外NK/T细胞淋巴瘤,鼻型(ENKTL)吉西他滨耐药中的分子机制。方法CCK-8法检测细胞增殖,Transwell小室法检测细胞的侵袭性,逐步增加吉西他滨浓度建立吉西他滨耐药YTS细胞系(YTS-gem),MTT法检测药物敏感性,Western blot法检测14-3-3ζ蛋白在YTS-gem细胞和YTS细胞中的表达差异。采用siRNA下调14-3-3ζ表达,对比下调前后YTS-gem细胞对吉西他滨敏感性的差异。Western blot法检测耐药相关蛋白在14-3-3ζ下调前后YTS-gem细胞中的表达差异。结果①与YTS细胞相比,14-3-3ζ在YTS-gem细胞中表达上调,差异有统计学意义(P<0.05);②与对照组相比,下调14-3-3ζ后YTS细胞和YTS-gem细胞的增殖和侵袭能力均明显受抑(P<0.05);③下调14-3-3ζ表达后,YTS-gem细胞对吉西他滨敏感性增加,差异有统计学意义(P<0.05);④下调14-3-3ζ表达后,YTS-gem细胞中促凋亡蛋白Bax水平显著升高,抗凋亡蛋白Bcl-2、caspase-3、cleaved caspase-3和Cyclin D1显著降低(P<0.05),而P-gp和p53表达水平的差异无统计学意义。结论14-3-3ζ在YTS-gem细胞中表达上调,14-3-3ζ促进细胞增殖和侵袭,14-3-3ζ蛋白通过抗凋亡途径诱导ENKTL对吉西他滨耐药。

Objective To explore the molecular mechanisms of 14-3-3ζ in gemcitabine resistance in extranodal NK/T-cell lymphoma,nasal type(ENKTL).Methods The effects of cell proliferation and invasion were detected by cell counting kit-8(CCK-8)assay and transwell assay.YTS cells were exposed to gradually increased concentrations of gemcitabine to establish gemcitabine-resistant YTS cells(YTS-gem)in vitro.14-3-3ζ specific siRNA lentiviral vector was transfected into YTS and YTS-gem cells to downregulate 14-3-3ζ expression,and stable transfected cell clones were screened.The protein expression was determined by Western blot.Results①14-3-3ζ expression was significantly up-regulated in gemcitabine resistant YTS-gem cells,comparing with that of YTS cells(P<0.05).②The results of CCK-8 and transwell assay showed that downregulation of 14-3-3ζ significantly reduced the cell proliferation and invasion abilities(P<0.05).③Downregulation of 14-3-3ζ could restore gemcitabine sensitivity in gemcitabine resistant YTS-gem cells(P<0.05).④Western blotting results showed that knockdown of 14-3-3ζ significantly upregulated pro-apoptotic Bax,and downregulated anti-apoptotic Bcl-2,Caspase-3,cleaved caspase-3,Cyclin D1 in gemcitabine-resistant YTS-gem cells(P<0.05).There was no significant difference in p53 ang P-gp expression levels.Conclusions 14-3-3ζ was upregulated in gemcitabine resistant YTS cells.Overexpression of 14-3-3ζ promoted cell proliferation and enhanced cell migration.14-3-3ζ contributed to gemcitabine resistance to ENKTL through anti-apoptosis.