Qin Chang-fu, Tian Xiao-dong, Hao Kun, Yang Yin-mo
Department of General Surgery, Peking University First Hospital, Beijing, China.
Zhonghua Yi Xue Za Zhi. 2012 Jul 10;92(26):1849-53.
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.
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 Gli1 were tested by real-time polymerase chain reaction (PCR). The antiproliferative effect was examined by the assays of colony formation and methyl thiazolyl tetrazolium (MTT). 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.
The RNAi technology with lentivirus 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 lower than that of simple EGFR RNAi (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.
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.
探讨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和凋亡的协同作用，并研究Hedgehog与EGFR信号通路的协同机制。
设计、合成靶向EGFR基因的RNA干扰序列并克隆至pFU-GW-RNAi载体，通过慢病毒转染人Panc-1细胞获得稳定转染细胞系。采用实时聚合酶链反应（PCR）检测Shh和Gli1的表达。通过集落形成实验和甲基噻唑基四氮唑（MTT）实验检测细胞增殖抑制作用。应用荧光激活细胞分选仪（FACS）检测各实验组细胞凋亡率。采用蛋白质免疫印迹法（Western blot）检测ERK和AKT的磷酸化水平。利用体内裸鼠成瘤模型检测生长抑制效果。
慢病毒介导的RNA干扰技术可抑制EGFR基因表达。RNA干扰沉默EGFR和Hedgehog信号通路后，人胰腺癌细胞对环杷明的化疗敏感性显著增加。环杷明的半数抑制浓度（IC50）从（2.978±0.336）降至（1.698±0.057）μmol/L（P<0.05）。联合处理组的早期凋亡率高达38.75%，显著高于RNA干扰沉默EGFR组（17.65%）和对照组（3.02%）（P<0.05）。肿瘤异种移植实验结果显示，联合处理组的肿瘤体积（394.8±87.5mm³）显著低于单纯EGFR RNA干扰组（594.7±86.1mm³）和单用环杷明处理组（771.3±82.9mm³）；联合处理在集落形成实验中也能产生明显的协同增殖抑制作用。RNA干扰沉默EGFR后，ERK和AKT的磷酸化水平较对照组显著降低。联合使用环杷明的联合处理组进一步降低。
RNA干扰沉默EGFR和Hedgehog信号通路可进一步抑制人胰腺癌细胞的体内外增殖并增加凋亡。Hh与EGFR信号通路的协同作用可能与ERK和AKT的磷酸化水平相关。
