Hedgehog-Gli信号通路调控EMT驱动前列腺癌干细胞特性形成的初步实验研究

Hedgehog-Gli pathway regulated epithelial mesenchymal transition induces stemness properties in prostate cancer cells

目的探索上皮间质转化EMT与肿瘤干细胞之间潜在联系及相关机制。方法应用免疫组织化学染色方法检测前列腺癌组织中肿瘤干细胞标志物表达情况。应用Q-PCR及Western blot方法检测分析ARCaPE和ARCaPM细胞中EMT及肿瘤干细胞相关标志物表达水平。应用细胞集落形成实验比较ARCaPE和ARCaPM细胞增殖能力。应用肿瘤克隆球形成实验检测细胞克隆形成和自我更新能力。应用GANT61封闭Hedgehog-Gli信号通路,分析Hedgehog-Gli通路阻断后ARCaPM细胞EMT及干细胞特性的变化。结果 CD44在前列腺癌中表达下调,且与分级相关,高级别(Gleason≥8)前列腺癌组织中下调更明显;Nanog在高级别前列腺癌中有表达增高现象;而CD133在前列腺良性增生中和前列腺癌中均为阴性染色。具有间质细胞样形态的ARCaPM细胞迁移侵袭能力较具有上皮细胞样形态的ARCaPE细胞明显增强,E-cadherin表达降低,N-cadherin、Vimentin、Snail表达增加。ARCaPM细胞中肿瘤干细胞分子标志物表达增高,其增殖及自我更新能力均明显增强。Gli1在ARCaPM细胞中表达增高,Gli抑制剂GANT-61可以有效抑制ARCaPM细胞EMT转化,降低其肿瘤干细胞分子标志物表达水平,抑制其增殖及自我更新能力。结论 Hedgehog-Gli信号通路调控的EMT可驱动前列腺癌细胞肿瘤干细胞特性形成。

Objective To explore the potential association between epithelial-mesenchymal transition process （EMT） and cancer stem cells （CSCs） and the possible mechanism. Methods The expressions of biomarkers in prostate cancer （PCa） tissues were detected with immunohistochemical method. The expressions of EMT and SCSc in ARCaPE and ARCaPM ceils were determined with quantitative polymerase chain reaction （Q-PCR） and Western blot. The proliferation ability of ARCaPE and ARCaPM ceils were compared with cell colony. Cell cloning and self-renewal were tested with clonal formation. After the Hedgehog-Glican pathway was blocked with GANT61, the changes of EMT of ARCaPM and the CSCs were analyzed. Results The expression of CD44 was down-regulated, in a grading-dependant manner, and the down-regulation was more obvious in PCa with Gleason≥8. The expression of Nanog was up-regulated. In both PCa and benign prostatic hyperplasia （BPH）, CD133 staining was negative. Compared with epithelial-phenotype ARCaPE cells, cell migration and invasion of mesenchymal-morphology ARCaPM cells showed increased migration capacity, decreased expression of E-cadherin, and increased expressions of N-cadherin, Vimentin, and Snail. ARCaPM showed high expression of CSC markers, proliferation, and self-renewal capac- ity. Gli transcription factor inhibitor GANT-61 could inhibit EMT and stemness properties of ARCaPM cells, decrease expression of CSC markers, and inhibit the proliferation and self-renewal capacity. Conclusion Hedgehog-Gli pathway regulated epithelial mesenchymal transition can induce sternness properties in prostate cancer cells, which provides new insights into clarifying the potential mechanisms of PCa oncogenesis.