靶向抑制EGFR联合阻断Hedgehog信号通路对胰腺癌细胞增殖的影响及机制

Effects and mechanisms of combined suppression of epidermal growth factor receptor and hedgehog signaling on proliferation in pancreatic cancer cells

目的观察靶向抑制表皮生长因子受体（EGFR）联合阻断Hedgehog信号通路对胰腺癌细胞增殖及凋亡的协同作用，探讨二者间的协同作用机制。方法设计并合成针对人EGFR基因特异性siRNA，构建慢病毒pFU-GW—RNAi载体，感染人胰腺癌Panc-1细胞，获得稳定转染细胞株。Real time PCR检测Shh和Glil的表达情况；克隆形成实验和MTI＂法检测细胞增殖情况；流式细胞技术检测细胞凋亡情况；Western印迹方法检测ERK和AKT磷酸化水平变化；裸鼠移植瘤模型检测移植瘤生长抑制情况。结果慢病毒介导的RNAi显著抑制Panc-1细胞EGFR的表达；靶向抑制EGFR后Panc-1细胞对环巴明的敏感性显著增加，细胞增殖能力下降，其半数抑制剂量（IC50）从（2．978±0．336）斗m0L／L下降到（1．698±0．057）μmoL／L，P〈0．05；联合处理组细胞早期凋亡比率高达38．75％，显著高于单独抑制EGFR组（17．65％）及对照组（3．02％），P〈0．05；裸鼠移植瘤实验结果显示联合处理组移植瘤体积（394．8±87．5）mm^3显著低于抑制EGFR组（594．7±86．1）mm^3和单独应用环巴明处理组（771．3±82．9）mm^3；克隆形成实验结果显示联合处理可以进一步抑制细胞增殖；抑制EGFR后胰腺癌细胞ERK和AKT磷酸化水平较对照组显著降低，联合应用环巴明处理后二者磷酸化水平均进一步下降。结论靶向抑制EGFR联合环巴明处理后胰腺癌细胞体内外增殖能力均进一步被抑制，细胞凋亡率进一步增加；其协同机制可能部分与调控ERK和AKT磷酸化水平相关。

Objective To explore the synergistic effects on proliferation and apoptosis by targeted suppression of epidermal growth factor receptor （EGFR） in combination with blockade of Hedgehog signaling pathways in pancreatic cancer cells and examine the synergistic mechanism of Hedgehog and EGFR signaling pathways. Methods The sequences of RNA interference targeting EGFR gene were designed, synthesized and cloned into the pFU-GW-RNAi vector. And a stable transfection cell line was obtained by transfecting the human Panc-1 cells with lentivirus. The expressions of Shh and Glil were tested by real-time polymerase chain reaction （PCR）. The antiproliferative effect was examined by the assays of colony formation and methyl thiazolyl tetrazolium （MTI＇）. Fluorescence activated cell sorter （FACS） was applied to assay the apoptotic rate in all experimental groups. Western blot was applied to detect the phosphorylation levels of ERK and AKT. In vivo nude mice tumorigenicity model was used to test the effect of growth inhibition. Results The RNAi technology with lentivirns could restrain the expression of EGFR gene. After the blocking of EGFR and Hedgehog signaling pathways by RNAi silencing, the chemosensitivity to cyclopamine significantly increased in human pancreatic cancer cells. The half-inhibitory concentration （IC 50） of cyclopamine declined from （ 2. 978 ± 0. 336 ） to （ 1. 698 ± 0. 057 ） μmol/L （ P 〈 0. 05 ）. The prophase apoptotic rate of co-treated group was as high as 38. 75% and it was significantly higher than the RNAi silencing EGFR （ 17.65% ） and control groups （ 3.02% ） （ P 〈 0. 05 ）. The results of tumor xenografts assay showed that the tumor volume of co-treated group （394. 8 ± 87. 5 mm^3） was significantly lowerthanthatofsimpleEGFRRNAi （ 594. 7 ± 86. 1 mm^3 ） and single cyclopamine treated group （ 771. 3 ±82.9 mm^3 ） ; the combination treatment could also produce obviously synergistic antiproliferative effect in colony formation assays. After RNAi silencing EGFR, the phosphorylation levels of ERK and AKT decreased significantly versus the control group. Further reduction was obtained with the combined use of cyclopamine in the co-treated group. Conclusion The blocking of EGFR and Hedgehog signaling pathways by RNAi silencing may further inhibit cell proliferation and increase apoptosis in vivo and in vitro in human pancreatic cancer cells. The synergism of Hh and EGFR signaling pathways may be correlated with the phosphorylation levels of ERK and AKT.