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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。
Histone deacetylase (HDACs) inhibitor sodium butyrate regulates cell differentiation, proliferation and inhibits tumorigenesis. Thioredoxin-interacting protein (TXNIP) regulates the redox balance and inhibits cell growth by negatively regulating the activity of thioredoxin. This study demonstrated that sodium butyrate can induce human non-small cell lung cancer A549 cell death by activating the expression of TXNIP that depends on the transcription factor NF-Y. MTT assay showed A549 cells treated with 5 mmol / L sodium butyrate for 72 h significantly induced death, and most of the cells died as apoptotic cells by flow cytometry. Expression microarray analysis showed that mRNA levels of TXNIP were significantly increased 30- to 50-fold in sodium butyrate-treated A549 cells. Real-time quantitative PCR, immunocytochemistry and Western blotting results further demonstrated that sodium butyrate significantly up-regulated TXNIP expression. Luciferase reporter assay demonstrated that sodium butyrate-stimulated intracellular reporter activity increased about 10-fold compared with control cells, suggesting that sodium butyrate activates the transcriptional activity of the TXNIP promoter. TXNIP promoter deletion mutation analysis showed that deletion of NF-Y-binding DNA sequence significantly reduced sodium butyrate TXNIP promoter activation, indicating NF-Y transcription factor involved in sodium butyrate-mediated TXNIP gene transcriptional activation. To analyze the localization and partial function of TXNIP in A549 cells, GFP-TXNIP fusion protein and its truncated mutant fusion protein were overexpressed in A549 cells. The results showed that wild-type and truncated mutant N-terminal 1-281aa Located in the nucleus, while deleting N-terminal 1-200aa, it located in the nucleus and cytoplasm, suggesting N-terminal 1-200aa can regulate the localization of the protein. However, sodium butyrate stimulated GFP-TXNIP, an undetectable expression, to be altered intracellularly. The above results indicate that sodium butyrate can induce the death of A549 cells by activating the transcription factor NF-YC-dependent TXNIP but can not change the localization of TXNIP protein in the cells. The above results also suggest that N-terminal 1-200aa of TXNIP may play a role in the regulation of the cellular localization of TXNIP. Whether sodium butyrate stimulates cell death induced by TXNIP expression is further demonstrated by studying the detailed regulatory mechanisms of TXNIP in cells by altering cellular oxidative stress.