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. Results: ①14-3-3ζ 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.
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。.
Keywords: Drug resistance; Gemcitabine; Lymphoma, extranodal NK/T-cell; Protein 14-3-3ζ.