丁酸钠至少部分通过NF-Y依赖的TXNIP表达诱导人肺癌细胞A549死亡  

Sodium Butyrate Induces Human Lung Cancer A549 Cell Death at Least Partly by Up-regulating NF-Y-dependent TXNIP Expression

作　　者：李秋霞[1] 吴娜娜[1] 吴建民[1] 肖小强[1] 

机构地区：[1]温州医科大学基因组研究院,浙江温州325000

出　　处：《中国生物化学与分子生物学报》2017年第5期478-486,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金青年基金(No.81302050)资助项目~~

摘　　要：组蛋白去乙酰化酶(HDACs)抑制剂丁酸钠调节细胞分化、增殖和抑制肿瘤发生。硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)通过负性调控硫氧还蛋白的活性,调控细胞内的氧化还原平衡,抑制细胞生长。本研究证明,丁酸钠可通过激活依赖于转录因子NF-Y的TXNIP表达,诱导人非小细胞肺癌细胞A549死亡。MTT法显示,5 mmol/L丁酸钠处理A549细胞72 h可显著诱导其死亡;流式细胞分析发现,其中大部分细胞以凋亡形式死亡。表达芯片分析表明,在丁酸钠处理的A549细胞中,TXNIP的mRNA水平显著提高30~50倍;实时定量PCR、免疫细胞化学和蛋白质印迹结果进一步证明,丁酸钠可显著上调TXNIP表达。荧光素酶报告基因分析证明,与对照细胞比较,丁酸钠刺激的细胞内报告酶活性可提高约10倍,提示丁酸钠可激活TXNIP启动子的转录活性。TXNIP启动子删除突变分析显示,删除NF-Y结合的DNA序列显著降低丁酸钠对TXNIP启动子的激活能力,表明NF-Y转录因子参与丁酸钠介导的TXNIP基因转录激活。为分析TXNIP在A549细胞中的定位和部分功能,在A549细胞中过表达GFP-TXNIP融合蛋白及其截短突变体融合蛋白;结果显示,野生型和保留N端1-281aa的截短突变体定位在细胞核,而删除N端1-200aa时,其定位在细胞核和细胞质,提示N端1-200aa可调节该蛋白质的定位。然而,丁酸钠刺激未发现表达的GFP-TXNIP在细胞内定位改变。以上结果表明,丁酸钠可通过激活转录因子NF-YC依赖的TXNIP激活,诱导A549细胞死亡,但不能改变TXNIP蛋白在细胞内的定位。上述结果还提示,TXNIP的N端1-200aa可能在调节TXNIP的细胞定位中发挥作用。是否丁酸钠刺激TXNIP表达导致的细胞死亡系通过改变细胞氧化压力,以及TXNIP在细胞中定位的详尽调节机制尚待进一步研究证明。Sodium butyrate, an inhibitor of histone deacetylases （HDACs） , regulates cell proliferation and differentiation and suppresses tumorigenesis. Thioredoxin interacting protein （TXNIP） interacts with thioredoxin and negatively regulates the activity of thioredoxin, thereby modulates the cellular reductionand oxidation status. Sodium butyrate could induce non-small cell lung cancer A549 cell death via activation of transcriptional factor NF-Y-dependent TXNIP expression. MTT method showed that treatment with 5 mmol/L sodium butyrate for 72 h could significantly induce A549 cell death, of which most cells died by apoptosis evidenced by flow eytometry. Mieroarray revealed that the level of TXNIP mRNA increased 30-50 folds in sodium butyrate-treated A549, which was further confirmed by RT-qPCR, immunoeytoehemistry and Western blot. Compared with control cells, the activity of reporter enzyme （lueiferase） increased about 10 folds in sodium butyrate-treated A549, indicating that sodium butyrate can activate the TXNIP promoter. Moreover, deletion of the NF-Y binding sequence on the TXNIP promoter markedly decreased sodium butyrate-stimulated activity of the TXNIP promoter, indicating that NF-Y is involved in sodium butyrate induced-TXNIP transcriptional activation. To understand the localization and biological function of TXNIP in A549, we overexpressed GFP-TXNIP fusion protein （wild type or its deletion mutants）, and showed the wild type and the mutant containing N-terminus 1-288 amino acid residues of TXNIP protein mainly localized in the nucleus, however, TXNIP mutant deleted 1- 200 amino acid sequence located in both plasma and nucleus, indicating that the 1-200 amino acid sequence of TXNIP N-terminus plays role in its cellular localization. We further found that sodium butyrate treatment did not change the localization of wild type and mutant of GFP-TXNIP in cells. In summary, sodium butyrate could induce lung cancer A549 cell death by activating NF-Y-dependent TXNIP expression, but cannot affect its l

关 键 词：非小细胞肺癌细胞A549 丁酸钠:硫氧还蛋白相互作用蛋白 核因子 

分 类 号：Q291[生物学—细胞生物学] R34[医药卫生—基础医学]