Background: The hedgehog signaling system (HHS) plays an important role in the regulation of cell proliferation and differentiation during the embryonic phases. However, little is known about the involvement of HHS in the malignant transformation of cells. This study aimed to detect the role of HHS in the malignant transformation of human bronchial epithelial (16HBE) cells.
Methods: In this study, two microfluidic chips were designed to investigate cigarette smoke extract (CSE)-induced malignant transformation of cells. Chip A contained a concentration gradient generator, while chip B had four cell chambers with a central channel. The 16HBE cells cultured in chip A were used to determine the optimal concentration of CSE for inducing malignant transformation. The 16HBE cells in chip B were cultured with 12.25% CSE (Group A), 12.25% CSE + 5 μmol/L cyclopamine (Group B), or normal complete medium as control for 8 months (Group C), to establish the in vitro lung inflammatory-cancer transformation model. The transformed cells were inoculated into 20 nude mice as cells alone (Group 1) or cells with cyclopamine (Group 2) for tumorigenesis testing. Expression of HHS proteins was detected by Western blot. Data were expressed as mean ± standard deviation. The t-test was used for paired samples, and the difference among groups was analyzed using a one-way analysis of variance.
Results: The optimal concentration of CSE was 12.25%. Expression of HHS proteins increased during the process of malignant transformation (Group B vs. Group A, F = 7.65, P < 0.05). After CSE exposure for 8 months, there were significant changes in cellular morphology, which allowed the transformed cells to grow into tumors in 40 days after being inoculated into nude mice. Cyclopamine could effectively depress the expression of HHS proteins (Group C vs. Group B, F = 6.47, P < 0.05) and prevent tumor growth in nude mice (Group 2 vs. Group 1, t = 31.59, P < 0.01).
Conclusions: The activity of HHS is upregulated during the CSE-induced malignant transformation of 16HBE cells. Cyclopamine can effectively depress expression of HHS proteins in vitro and prevent tumor growth of the transformed cells in vivo.
抑制Hedgehog信号转导通路能够减弱微流控芯片中烟雾对16HBE细胞的恶性分化诱导作用摘要背景：Hedgehog信号系统(HHS) 在胚胎期对于调节细胞增殖和分化起到重要作用。然而，人们对于HHS在细胞恶性转化中的作用还知之甚少。本研究的目的在于检测HHS在人支气管上皮细胞（16HBE）恶性转化中的作用。 方法：在本研究中，设计了两个微流控芯片来完成香烟提取物(cigarette smoke extract，CSE)诱导的细胞恶性转化研究。芯片A含有一个浓度梯度发生器 (concentration gradient generator) ，芯片B含有4个细胞培养池和一个中央通道。芯片A中的16HBE细胞用于确定最佳CSE浓度，芯片B中培养的16HBE细胞用于(1) A组: 正常全培养基，(2) B组：12.25% CSE， (3) C组：12.25% CSE + 5μmol/L 环巴胺（cyclopamine） 培养8个月，建立肺部炎-癌转化体外模型。转化后的细胞接种到20只裸鼠中(1组)或细胞+环巴胺 (2 组)检测成瘤性。成对样本比较使用t检验，多组间差异采用方差分析。 结果：结果表明最佳CSE浓度为12.25%。HHS蛋白的表达在恶性转化过程中增加(B组 vs. A组, F=7.65, P＜0.05)。CSE处理8个月后，细胞形态发生明显变化，并且在植入裸鼠40天内能够长成肿块。环巴胺能够有效抑制HHS蛋白表达(B组 vs. A组，F=6.47, P＜0.05) 并抑制肿瘤生长(2组 vs. 1组, t=31.59, P＜0.01)。 结论：HHS活性在CSE诱导的细胞恶性转化过程中表达上调。环巴胺能够有效抑制HHS蛋白表达并阻止转化细胞长成肿瘤。.
Keywords: Hedgehog Signaling System; Lung Cancer; Malignant Transformation; Microfluidic Chip.