Huang Chao, Liu Haosheng, Yang Yidian, He Yue, Shen Weizeng
Department of Traditional Chinese Medicine, The Second Affiliated Hospital of Shenzhen University (People's Hospital of Shenzhen Baoan District), Shenzhen, 518100, China.
Heliyon. 2024 Aug 12;10(16):e36059. doi: 10.1016/j.heliyon.2024.e36059. eCollection 2024 Aug 30.
To explore the mechanisms of the TGF-β1/Smad and NF-κB pathways in the effect of berberine (BBR) on colon cancer epithelial-mesenchymal transition (EMT) and their regulatory relationships with microRNAs (miRNAs).
TGF-β1 was used to induce EMT in normal colon epithelial HCoEpiC cells and colon cancer HT29 cells in vitro. After BBR intervention, the expression of EMT-related markers and the major molecules involved in the TGF-β1/Smad and NF-κB pathways were detected via western blotting. Cell migration was detected via wound healing assays. SMAD2 and NF-κB p65 were overexpressed and transfected into cells, and the inhibitors SB431542 and BAY 11-7082 were added to block the TGF-β1/Smad and NF-κB pathways, respectively. The mRNA expression levels of related microRNA genes were detected by using RT‒PCR.
Treatment with 10 ng/ml TGF-β1 for 72 h significantly induced EMT in HCoEpiC and HT29 cells, which was repressed by BBR. BBR significantly inhibited the TGF-β1-induced migration of HCoEpiC and HT29 cells and the TGF-β1-promoted expression of p-Smad2/3, NF-κB p65, and p-IκBα. Compared to those in the group treated with TGF-β1, the expression of NF-κB p65 and p-Smad2 in the group treated with NF-κB pathway inhibitor BAY 11-7082 was decreased ( < 0.05), and TGF-β1 signalling inhibitor SB431542 significantly reduced the expression of NF-κB p65 ( < 0.05). Overexpression of NF-κB p65 and SMAD2 in HT29 cells decreased the expression of -cadherin and caused a relative increase in N-cadherin. BBR mediated the expression profile of microRNAs in TGF-β1-induced HCoEpiC cells, but this pattern differed from that in HT29 cells. SB431542 and BAY 11-7082 significantly reduced the mRNA level of miR-1269a in HCoEpiC and HT29 cells ( < 0.05). Overexpressed NF-κB p65 and SMAD2 increased the mRNA level of miR-1269a in both cell lines; however, this increase was significantly lower than that in the TGF-β1 treatment group ( < 0.05).
BBR can significantly inhibit TGF-β1-induced EMT in normal and cancerous colon epithelial cells through the inhibition of the TGF-β1/Smad and NF-κB p65 pathways. TGF-β1/Smads can promote the NF-κB p65 pathway, which is a common target of miR-1269a, and can partially regulate the expression of miR-1269a.
探讨转化生长因子-β1（TGF-β1）/Smad和核因子-κB（NF-κB）信号通路在小檗碱（BBR）影响结肠癌上皮-间质转化（EMT）中的作用机制及其与微小RNA（miRNA）的调控关系。
采用TGF-β1体外诱导正常结肠上皮HCoEpiC细胞和结肠癌HT29细胞发生EMT。BBR干预后，通过蛋白质免疫印迹法检测EMT相关标志物以及TGF-β1/Smad和NF-κB信号通路中主要分子的表达。采用划痕实验检测细胞迁移能力。分别过表达SMAD2和NF-κB p65并转染至细胞中，同时加入抑制剂SB431542和BAY 11-7082分别阻断TGF-β1/Smad和NF-κB信号通路。采用逆转录-聚合酶链反应（RT-PCR）检测相关miRNA基因的mRNA表达水平。
10 ng/ml TGF-β1处理72 h可显著诱导HCoEpiC和HT29细胞发生EMT，而BBR可抑制该过程。BBR显著抑制TGF-β1诱导的HCoEpiC和HT29细胞迁移以及TGF-β1促进的p-Smad2/3、NF-κB p65和p-IκBα表达。与TGF-β1处理组相比，NF-κB信号通路抑制剂BAY 11-7082处理组中NF-κB p65和p-Smad2的表达降低（P<0.05），TGF-β1信号抑制剂SB431542显著降低NF-κB p65的表达（P<0.05）。HT29细胞中NF-κB p65和SMAD2过表达可降低E-钙黏蛋白的表达并使N-钙黏蛋白相对增加。BBR可介导TGF-β1诱导的HCoEpiC细胞中miRNA的表达谱，但与HT29细胞中的模式不同。SB431542和BAY 11-7082显著降低HCoEpiC和HT29细胞中miR-1269a的mRNA水平（P<0.05）。过表达的NF-κB p65和SMAD2可增加两种细胞系中miR-1269a的mRNA水平；然而，这种增加显著低于TGF-β1处理组（P<0.05）。
BBR可通过抑制TGF-β1/Smad和NF-κB p65信号通路，显著抑制TGF-β1诱导的正常和癌性结肠上皮细胞EMT。TGF-β1/Smads可促进NF-κB p65信号通路，该通路是miR-1269a的共同靶点，并可部分调节miR-1269a的表达。
