NF-κB和TGF-β/Smads通路交互调控TAF诱导肠上皮EMT相关机制及中药单体干预研究

Phenotypic transition of stromal cells is one of the critical mechanisms in tumor microenvironment remodeling. Tumor-associated fibroblasts (TAFs) play an important role in the induction of epithelial EMT transition. Previous studies have shown that TAF-like CCD-18Co cells induced EMT in colonic epithelial cell line HCoEpiC, and the EMT was suppressed by berberine and inhibitors of NF-κB and TGF-β/Smads pathway at different extent. Thus, we hypothesize that "①interaction of NF-κB and TGF-β/Smads pathway promotes the expression of EMT transcription factors (EMT-TFs), regulating the CCD-18Co-induced EMT in HCoEpiCs; ②berberine mediates the interaction to inhibit downstream EMT-TFs, suppressing the above EMT". To verify this hypothesis, we will use an in vivo colon cancer rat model, and HCoEpiC cells to establish an in vitro model of CCD-18Co-induced EMT, as well as corresponding inhibitors, berberine, RNAi and overexpression transfection of genes are adopted. This study will contribute to providing experimental evidence for clarifying the mechanisms of colon epithelial phenotype transition induced by TAFs and for revealing the mechanisms of anti-tumor microenvironment remodeling by Traditional Chinese Medicine.

基质细胞表型转变是肿瘤微环境重塑的重要机制之一。肿瘤相关成纤维细胞（TAFs）在上皮表型EMT的诱发中扮演重要角色。前期研究表明TAF样的CCD-18Co细胞可诱导结肠上皮HCoEpiCs细胞发生EMT，并可受NF-κB、TGF-β/Smads通路抑制剂和小檗碱不同程度的抑制。因此我们假设“①NF-κB与TGF-β/Smads通路交互促进EMT转录因子（TFs）表达，从而调节CCD-18Co诱导HCoEpiCs发生EMT；②小檗碱调控该交互作用从而抑制下游EMT-TFs表达来干预上述EMT效应”。拟通过结肠癌大鼠体内模型以及HCoEpiCs细胞为对象构建CCD-18Co诱导EMT发生的体外模型，加入相应抑制剂、小檗碱以及应用RNAi、过表达转染技术来验证该假说。本研究有助于阐明TAFs促肠上皮表型转变机制及揭示中医药抗肿瘤微环境重塑机理提供实验依据。

基质细胞表型转变是肿瘤微环境重塑的重要机制之一。恶性肿瘤细胞在上皮表型EMT的诱发中扮演重要角色。前期研究表明可分泌细胞因子TGF-β1的TAF样的结肠成纤维CCD-18Co细胞可诱导结肠上皮HCoEpiCs细胞发生EMT，并可受NF-κB、TGF-β/Smads通路抑制剂和小檗碱不同程度的抑制。因此我们假设“①NF-κB与TGF-β/Smads通路交互促进EMT转录因子（TFs）表达，从而调节结肠癌细胞和正常结肠上皮细胞发生EMT；②小檗碱调控该交互作用从而抑制下游EMT-TFs表达来干预上述EMT效应”。本研究通过结肠癌细胞系和乳鼠体内模型以及HT29细胞、HCoEpiCs细胞为对象构建TGF-β1诱导EMT发生的体外模型，加入相应抑制剂和小檗碱来验证该假说。结果显示TGF-β1可显著诱导结肠癌细胞和正常结肠上皮细胞发生EMT改变，加入小檗碱可明显抑制这种EMT改变并抑制细胞增殖，小檗碱可明显抑制TGF-β/Smads通路和NF-κB p65通路，同时小檗碱还可改变小鼠体内肠道菌群内环境，这与相关表观遗传调节因子的调控有关。本研究结果表明小檗碱可通过调控TGF-β/Smads通路和NF-κB p65通路和表观遗传相关因子及肠道菌群来抑制结肠上皮细胞的EMT和细胞生长，本项目有助于阐明TGF-β促结肠上皮细胞表型转变机制及揭示中医药抗肿瘤微环境重塑机理提供实验依据。