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目的:探讨舒尼替尼通过NF-κB信号通路诱导肝癌HepG2细胞表达自然杀伤细胞2族成员D配体(natural killer group 2 member D ligands,NKG2DLs)的分子机制。方法:常规体外培养HepG2细胞,单细胞凝胶电泳检测1μmol/L舒尼替尼处理HepG2细胞24 h前后DNA损伤情况,实时荧光定量PCR检测药物处理前后细胞DNA损伤修复分子mRNA的表达,Western blotting检测分别以NF-κB激动剂和抑制剂处理HepG2细胞前后NKG2DLs蛋白表达及IKKα和IκBα表达情况。结果:舒尼替尼药物处理后,HepG2细胞均发生不同程度DNA损伤;且AP-1、ATM、ATR mRNA表达水平明显升高,而CHK1、CHK2、GSK3βmRNA表达水平明显降低;不同处理组间DNA损伤修复相关信号分子mRNA表达有显著差异(F=61.242,P=0.000)。NF-κB转录活性抑制剂JSH-23可降低HepG2细胞NKG2DLs蛋白表达量,而NF-κB转录活性激动剂TNF-α、PMA均可增加HepG2细胞NKG2DLs蛋白表达量(F=15.043,P=0.000);舒尼替尼处理肿瘤细胞后NF-κB的抑制分子IKKα被抑制,而激活分子IκBα被激活。结论:舒尼替尼可通过DNA损伤修复分子激活NF-κB旁路途径诱导肿瘤细胞表达NKG2DLs。
OBJECTIVE: To investigate the molecular mechanism of sunitinib-induced natural killer group 2 member D ligands (NKG2DLs) expression in HepG2 hepatoma cells through NF-κB signaling pathway. Methods: HepG2 cells were cultured in vitro. The DNA damage of HepG2 cells treated with 1 μmol / L of sunitinib for 24 h was detected by single cell gel electrophoresis. The expression of DNA damage repair mRNA was detected by real-time fluorescence quantitative PCR. Western blotting The expression of NKG2DLs and the expression of IKKα and IκBα in HepG2 cells treated with NF-κB agonists and inhibitors were detected. Results: After treated with sunitinib, HepG2 cells had different levels of DNA damage. The expression of AP-1, ATM and ATR mRNA was significantly increased, while the expressions of CHK1, CHK2 and GSK3β mRNA were significantly decreased. There was a significant difference in mRNA expression of injury-repair related signaling molecules (F = 61.242, P = 0.000). NF-κB transcriptional activity inhibitor JSH-23 decreased the expression of NKG2DLs in HepG2 cells, whereas NF-κB transcriptional activators TNF-α and PMA increased the expression of NKG2DLs in HepG2 cells (F = 15.043, P = 0.000) The inhibitory molecule IKKα of NF-κB was inhibited after sunitinib treatment of tumor cells, while the activation molecule IκBα was activated. Conclusion: Sunitinib can induce NKG2DLs expression in tumor cells through NF-κB pathway activated by DNA damage repair molecule.