摘要
目的观察特异性阻断hedgehog（Hh）信号通路对胰腺癌Panc一1细胞上皮间质转化（EMT）过程的影响，探讨其分子机制。方法设计并合成针对Smoothened（SMO）基因特异性的RNA干扰靶点，运用慢病毒介导的RNA干扰技术沉默SMO基因表达以阻断人胰腺癌Panc-1细胞Hh信号通路。应用Matrigel／Transwell法检测阻断Hh信号通路后胰腺癌细胞侵袭能力的变化，应用实时PCR及Western印迹方法检测阻断Hh通路后Panc一1细胞EMT标志物E—cadhefin、N—cadhefin、B．catenin、Vimentin、Fibronectin等的表达变化；并检测EMT调控因子Snail、Slug等的表达变化。结果稳定干扰SMO基因表达阻断Hh信号通路后，人胰腺癌Panc-1细胞侵袭能力明显降低，P=0．020；上皮表型标志物E．cadherin表达明显上调，而间质表型Vimentin、Fibronectin、N—cadhenn等的表达水平显著下调，P值分别为0．020、0．001、0．031和0．022；与对照组相比，SMO干扰组细胞中转录因子Snail、Slug的表达水平均显著下调，P值分别为0．018和0．016。结论应用慢病毒介导的RNA干扰技术特异性沉默SMO基因阻断人胰腺癌细胞中Hh通路活性，可显著抑制Panc-1细胞EMT过程，其机制与下调转录因子Snail及Slug表达有关。
Objective To explore the blocking effects of hedgehog signaling pathway on the processes of cell migration, invasion and epithelial-mesenchymal transition （EMT） in human pancreatic cancer cells and elucidate its possible mechanisms. Methods The lentiviral expression vector for RNA interference of human Smoothened （SMO） gene was constructed to silence the expression of SMO. And RNAi against SMO was used to suppress the hedgehog signaling pathway in human pancreatic cancer Panc-1 cells. The in vitro invasion capacity in Panc-1 cells was assessed by Matrigel/Transwell chamber assay. Real-time PCR （polymerase chain reaction） and Western blot were used to detect the expressions of such EMT markers as E-cadherin, N-cadherin, ^-catenin, vimentin and fibronectin and such transcription factors as Snail, Slug, Twistl and Sipl. Results The stable interference of SMO could suppress the hedgehog signaling activity in Panc-1 cells. The inhibition of hedgehog signaling reduced the in vitro invasion capacity significantly in Panc-1 cells. The expression of E-cadherin significantly increased while N-cadherin, vimentin and fibronectin were significantly down-regulated in the RNAi group. Compared to the control group, the expressions of Snail and Slug were significantly reduced in the SMO knock-down group. Conclusion The inhibition of hedgehog signaling pathway reduces the in vitro invasion capacity in human pancreatic cancer cells. And the EMT process is significantly suppressed. The mechanism is partially correlated with the down-regnlations of Snail and Slug.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2011年第44期3103-3106,共4页
National Medical Journal of China
基金
国家自然科学基金（30972897）
关键词
胰腺肿瘤
肿瘤
腺和上皮
HEDGEHOG
上皮间质转化
Pancreatic neoplasms
Neoplasms, glandular and epithelial
Hedgehog
Epithelial-mesenchymal transition
