摘要目的 观察胃癌细胞株中泛素连接酶干细胞因子(SCF)对AT丰富结合域1A基因(ARID1A)表达的调控作用.方法 通过DNA损伤诱导剂处理胃癌细胞株,分别用蛋白质合成抑制剂CHX、蛋白酶体抑制剂MG132和环氧甲酮四肽蛋白酶体抑制剂(epoxomicin)处理细胞,利用NAE酶抑制剂MLN4924干扰泛素化,反转录-聚合酶链反应(RT-PCR)和Western blot检测不同方法处理后ARID1A mRNA和蛋白的表达,采用Lipofectamine 2000转染Flag-ARID 1A和显性抑制Cullin,Western blot和免疫共沉淀法检测泛素化的ARID1A以及内源性的S期激酶相关蛋白1(SKP1)和Cullin1蛋白表达.结果 在胃癌细胞系NCI-N87和AGS中,用0.5、1.0、2.0、4.0 μg/ml VP16处理24 h后ARID1A的相对蛋白表达量分别为0.803±0.037、0.717±0.056、0.271±0.072、0.301±0.034和0.741±0.023、0.657±0.048、0.235±0.044、0.278±0.041,提示DNA损伤后出现ARID1A表达下调是一种普遍现象(P＜0.05).DNA损伤诱导剂VP16影响ARID1 A的表达稳定是由其导致的ARID1A降解所致,而蛋白酶体抑制剂可以抑制VP16导致的蛋白降解并观察到泛素化的ARID1A.MLN4924可以有效地阻断VP16诱导的ARID1A降解,而用VP16诱导DNA损伤应答后,只有转染入DN-Cullin1组能够稳定表达ARID1A.293T细胞中转染入Flag-ARID1A后,在沉淀出的Flag-ARID1A复合物中可以检测出内源性SKP1和Cullin1蛋白的表达.结论 SCF连接酶是DNA损伤应答后ARID1A降解所必需的,胃癌细胞DNA损伤后ARID1A会被SCF连接酶迅速识别并降解.

abstractsObjective To investigate the regulation of AT-rich interaction domain 1A(ARID1A) via Cullin-S-phase kinase-associated protein 1 (SKP1)-F-box E3 ligase (SCF) ubiquitin ligase in gastric cancer cells.Methods Gastric cancer cells were treated with DNA damage response inducing reagent,protein synthesis inhibitor cycloheximide (CHX),proteasome inhibitor MG132 and epoxomicin.Using the MLN4924,a inhibitor of NEDD8-activating enzyme (NAE),to block ubiquitination,the expression of ARID1A mRNA and protein after different treatment were detected by real-time quantitative polymerase chain reaction (Real-time PCR) analysis and Western blotting,Flag-ARID1A and dominant negative (DN) Cullins were transfected with Lipofectamine 2000,the ubiquitinated ARID1A protein,endogenous Cullin1 and SKP1 were detected by Western blotting and immunoprecipitation.Results The relative expression of ARID1A treated with 0.5,1.0,2.0,4.0 μg/ml VP16 after 24 hours were 0.803 ± 0.037,0.717 ± 0.056,0.271 ± 0.072,0.301 ± 0.034 in NCI-N87 and were 0.741 ± 0.023,0.657 ± 0.048,0.235 ± 0.044,0.278 ± 0.041 in AGS,respectively,then the result reveal that the decrease of ARID1A protein is a common event during DNA damage of gastric cancer cells (P ＜0.05).The stability of ARID1A protein was influenced with DNA damage response inducing reagent VP16,which could cause the degradation of ARID1 A,Moreover,the proteasome inhibitor could efficiently prevent VP16-induced ARID1A degradation,and ubiquitinated ARID1A protein was appeared in response to VP16 treatment.The MLN4924 could efficiently block VP16-induced ARID1A degradation,only DN-Cullin1 could significantly stabilize ARID1A during DNA damage response induced by VP16.293T cells were transfected with Flag-ARID1A,and then we found that both endogenous Cullin1 and SKP1 were detected in the precipitated Flag-ARID1A complex.Conclusion Cullin-SKP1-F-box E3 ligase was required for the efficient degradation of ARID1A during DNA damage response,and ARID1A was rapidly ubiquitinated and degradated in response to DNA damage by SCF E3 ligase in gastric cancer cells.