[Construction of IL-6/STAT3 reporter gene system for screening and validation of traditional Chinese medicine monomers inhibiting IL-6/STAT3 signaling pathway].

It is important to find monomers of traditional Chinese medicine that can inhibit the activation of IL-6/STAT3 signaling pathway to suppress the growth and deterioration of tumors. A novel expression vector containing STAT3 enhancer sequence and NanoLuc (NLuc) reporter gene sequence was constructed by gene recombination technology, then a cell line expressing NLuc luciferase and regulated by STAT3 was further established. The cell line was used to quantitatively detect the regulatory effects of various traditional Chinese medicine monomers on the IL-6/STAT3 signal pathway. The effects of the traditional Chinese medicine monomers inhibiting the IL-6/STAT3 signal pathway were verified by Western blotting immunoassay and Real-time PCR analysis. By enzyme digestion and DNA sequencing analysis, it showed that the reporter gene expression vector pQCXIP-STAT3-Nluc was constructed successfully. The addition of Interleukin-6 (IL-6), a stimulator of STAT3 transcription factor, to the constructed cell line specifically enhanced the luciferase enzymatic reaction in a dose dependent manner. These results demonstrated that the cell line expressing NLuc luciferase and regulated by STAT3 has been constructed successfully. We used the the cell line screened out dendrobiine and tetrandrine which could significantly inhibit IL-6/STAT3 signal pathway and down-regulated the expression of Bcl-2 and Bcl-x in a dose-dependent manner. In conclusion, we have constructed an efficient reporter gene system to detect the transcriptional activity of STAT3, by which the Chinese medicine monomers inhibiting IL-6/STAT3 signaling pathway were successfully screened out, which has some potential theoretical and practical values.寻找可抑制 IL-6/STAT3 信号通路的活化从而抑制肿瘤的生长和恶化的中药单体化合物具有重要意义及发展前景。文中通过基因重组技术构建出一种含有STAT3 增强子序列和 NanoLuc (NLuc) 报告基因序列的新表达载体，并进一步建立受STAT3 调控并稳定表达NLuc 荧光素酶的细胞系，利用该细胞系定量检测多种中药单体化合物对IL-6/STAT3 信号通路的调控作用，并对抑制IL-6/STAT3 信号通路的中药单体的效果进行验证。酶切鉴定及测序结果表明报告基因表达载体pQCXIP-STAT3-NLuc 构建成功。STAT3 转录因子的刺激物白细胞介素-6(IL-6) 作用于所构建的稳定表达NLuc 的细胞系后出现特异性荧光素酶反应，且作用效果呈良好的剂量依赖性，表明受STAT3 调控稳定表达NLuc 荧光素酶的细胞系构建成功。Western blotting 及Real-time PCR 实验结果表明所筛选的中药单体化合物石斛碱及粉防己碱可抑制IL-6/STAT3 信号通路并显著下调其下游基因Bcl-2 及Bcl-x 的表达，且作用呈剂量依赖性。综上所述，文中构建了可高效检测STAT3 转录活性的报告基因系统，并利用该系统成功地筛选出可抑制IL-6/STAT3 信号通路的中药单体化合物，具有一定的理论和应用价值。.