【摘要】 目的 探讨脂肪量和肥胖相关蛋白(obesity-associated protein, FTO)介导的X-框结合蛋白1(X-box binding protein 1,XBP1)N6腺苷酸甲基化(N6-methyladenosine, m6A)去甲基化修饰在姜黄素诱导的Huh7肝癌细胞凋亡中的作用及机制。方法 (1)采用不同浓度(0、5、10、20、40、60μmol/L)姜黄素处理Huh7细胞48 h, CCK-8检测细胞存活率；流式细胞术检测细胞凋亡水平；Western blot检测细胞中FTO、XBP1及凋亡相关蛋白Bax、Bcl-2、cleaved Caspase-3表达水平；甲基化RNA免疫共沉淀法检测各组细胞中XBP1 RNA m6A甲基化水平。(2)将FTO干扰片段(si-FTO)及其阴性对照(si-NC)、FTO过表达质粒(oe-FTO)及其阴性对照(Vector)转染至Huh7细胞中，再联合20μmol/L姜黄素或160μmol/L甲基化抑制剂3-脱氮腺苷(3-deazaadenosine, 3-DAA)处理48 h, qRT-PCR法检测细胞中FTO和XBP1 mRNA表达水平；流式细胞术检测细胞凋亡水平；甲基化RNA免疫共沉淀法检测细胞中XBP1 RNA m6A甲基化水平；放线菌素D实验检测细胞XBP1 mRNA半衰期；Western blot检测细胞中FTO、XBP1、cleaved Caspase-3等蛋白表达水平。结果 不同浓度姜黄素干预可显著抑制Huh7细胞存活，诱导细胞凋亡(均P<0.05);同时，上调细胞中Bax、cleaved Caspase-3蛋白表达水平和XBP1 RNA m6A甲基化水平，下调Bcl-2、FTO和XBP1蛋白表达水平(均P<0.05)。沉默FTO基因可诱导Huh7细胞凋亡，提高细胞中XBP1 RNA m6A甲基化水平，缩短XBP1 mRNA半衰期，下调XBP1 mRNA和蛋白表达水平(均P<0.05)。然而，3-DAA干预显著逆转FTO基因沉默介导XBP1 mRNA m6A修饰诱导的Huh7细胞凋亡；同时，过表达FTO显著逆转姜黄素对Huh7细胞凋亡的诱导作用。结论 姜黄素可通过下调FTO表达，抑制XBP1 m6A去甲基化，进而促进XBP1 mRNA和蛋白降解，诱导肝癌细胞凋亡。更多还原

【Abstract】 Objective To investigate the role and mechanism of fat mass and obesity-associated protein(FTO)-mediated N6-methyladenosine(m6A)demethylation of X-box binding protein 1(XBP1)in curcurin-induced apoptosis of Huh7 liver cancer cells.Methods(1)Huh7 cells were treated with various concentrations of curcumin(0,5,10,20,40,60 μmol/L)for 48 h, and the cell viability was assessed using the CCK-8 assay.Apoptosis was analyzed by flow cytometry.The protein expression levels of FTO,XBP1,Bax, Bcl-2 and cleaved Caspase-3 were determined by Western blot analysis.The methylation level of m6A in XBP1 RNA was measured by methylated-RNA immunoprecipitation.(2)Huh7 cells were transfected with FTO interference fragment(si-FTO)or its negative control(si-NC),as well as FTO overexpression plasmid(oe-FTO)or its negative control(Vector).Subsequently, the cells were treated with 20 μmol/L curcumin or 160 μmol/L of the methylation inhibitor 3-deazaadenosine(3-DAA)for 48 h.The mRNA expression levels of FTO and XBP1 were quantified by qRT-PCR.Apoptosis was evaluated by flow cytometry.The methylation level of m6A in XBP1 RNA was detected by methylated-RNA immunoprecipitation.The half-life of XBP1 mRNA was determined by actinomycin D treatment.The protein expression levels of FTO,XBP1,and cleaved Caspase-3 were analyzed by Western blot.Results Treatment with various concentrations of curcumin significantly suppressed the proliferation and survival of Huh7 cells while inducing apoptosis(all P<0.05).Concurrently, the protein expression levels of Bax and cleaved Caspase-3,as well as the methylation level of XBP1 RNA m6A in Huh7 cells, were up-regulated, whereas the protein expression levels of Bcl-2,FTO and XBP1 were down-regulated(all P<0.05).Silencing of the FTO gene promoted apoptosis in Huh7 cells, increased the methylation level of XBP1 RNA m6A,shortened the half-life of XBP1 mRNA,and down-regulated the expression levels of XBP1 mRNA and protein(all P<0.05).However, intervention with 3-DAA markedly reversed the apoptosis of Huh7 cells induced by FTO gene silencing-mediated XBP1 mRNA m6A modification.Additionally, overexpression of FTO significantly counteracted the pro-apoptotic effect of curcumin on Huh7 cells.Conclusion Curcumin down-regulates the expression of FTO,inhibits the demethylation of XBP1 m6A,promotes the degradation of XBP1 mRNA and protein, and induces apoptosis in liver cancer cells.更多还原