Inhibition of TNF-alpha gene expression and bioactivity by site-specific transcription factor-binding oligonucleotides.
Inhibition of TNF-alpha gene expression and bioactivity by site-specific transcription factor-binding oligonucleotides.
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通过位点特异性转录因子结合寡核苷酸抑制 TNF-α 基因表达和生物活性。
DOI：
10.1152/ajplung.00134.2002
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Rojanasakul,Yon
中科院分区：
文献类型：
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作者：
Ye,Jianping;Wang,Liying;Zhang,Xiaoying;Tantishaiyakul,Vimon;Rojanasakul,Yon
本研究探讨了核因子结合寡核苷酸(ON)对肿瘤坏死因子-α基因的转录失活及其对小鼠肺部炎症反应的影响。采用聚合酶链式反应、基因突变和凝胶移位分析鉴定核因子结合和脂多糖反式激活肿瘤坏死因子-α基因所需的特异性顺式作用元件。内毒素诱导的肿瘤坏死因子-α需要多个结合位点的转录激活，包括−850(κ1)、−655(κ2)和−510(κ3)，而−210(κ4)没有作用。最大诱导性与κ3位点的激活有关。针对该位点的序列特异性双链对脂多糖诱导的肿瘤坏死因子-α活性的抑制作用最强。用小鼠肺部炎症模型研究了ON对肿瘤坏死因子-α生物活性的抑制作用。但不能抑制内毒素诱导的炎性中性粒细胞内流和肺细胞产生肿瘤坏死因子-α。在这个模型中，On的有效抑制被证明需要一种脂质体来有效地在细胞内传递On。综上所述，我们的结果表明，利用竞争核因子与肿瘤坏死因子α基因启动子结合的ON可以实现肿瘤坏死因子α基因的转录失活。这种基因抑制方法可以作为一种研究工具，或作为一种潜在的治疗手段，用于病因学依赖于基因异常表达的疾病。
The present study investigated transcriptional inactivation of TNF-α gene by nuclear factor-binding oligonucleotides (ON) and their effects on pulmonary inflammatory responses in mice. PCR-based gene mutation and gel shift assays were used to identify specificcis-acting elements necessary for nuclear factor binding and transactivation of TNF-α gene by lipopolysaccharide (LPS). LPS inducibility of TNF-α was shown to require transcriptional activation by NF-κB at multiple binding sites, including the −850 (κ1), −655 (κ2), and −510 (κ3) sites, whereas the −210 (κ4) site had no effect. Maximum inducibility was associated with the activation of κ3 site. The sequence-specific, double-stranded ON targeting this site was most effective in inhibiting TNF-α activity induced by LPS. The inhibitory effect of ON on TNF-α bioactivity was also investigated using a murine lung inflammation model. Pretreatment of mice with ON, but not its mutated sequence, inhibited LPS-induced inflammatory neutrophil influx and TNF-α production by lung cells. Effective inhibition by ON in this model was shown to require a liposomal agent for efficient cellular delivery of the ON. Together, our results indicate that transcriptional inactivation of TNF-α gene can be achieved by using ON that compete for nuclear factor binding to TNF-α gene promoter. This gene inhibition approach may be used as a research tool or as potential therapeutic modality for diseases with etiology dependent on aberrant gene expression.