摘要
目的探讨前列腺癌上皮细胞间质转化现象（EMT）是否在肿瘤干细胞（CSC）水平发生。方法（1）将脂质体2000系统将缺氧诱导因子1仅（HIF一1d）cDNA分别转染人前列腺癌细胞株LNCaP中的干细胞亚群LNCaP／SP和非干细胞亚群LNCaP／NSP，G418筛选稳定表达HIF-1a的抗性克隆。（2）采用Western印迹法鉴定HIF-1a及两种受其调控的下游基因的蛋白表达。同时检测细胞转染前后EMT指标蛋白的表达变化。（3）应用Transwell和四甲基偶氮唑盐（MTI＂）技术验证两种转染后细胞的体外侵袭和增殖能力的变化。（4）将两种转染后细胞裸鼠皮下致瘤，观察致瘤率、转移率及瘤体体积的差异。结果Western印迹法证实，与LNCaP／SP相比，LNCaWHIF-1a／SP发生了典型的EMT：上皮型钙黏蛋白（E．cadherin）和细胞角蛋白18（CKl8）表达缺失，同时波形纤维蛋白（vimentin）、神经型钙黏蛋白（N—cadhefin）、纤维连接蛋白（fibronectin）、组织蛋白酶D（cathepsinD）、基质金属蛋白酶2（MMP-2）和尿激酶型纤溶酶原激活物受体（uPAR）反常表达；另外，与LNCaP／NSP相比，LNCaP／HIF．1a／NSP发生了部分EMT转化（CKl8表达缺失，同时vimentin和cathepsinD反常表达）。与LNCaP／HIF-1a／NSP相比，LNCaP／HIF．1a/sP的体外增殖和侵袭能力更强大。LNCaWHIF-1w／sP不仅在裸鼠体内的皮下致瘤率（80％比53％，P〈0．05）和骨转移率（40％比0，P〈0.01）显著高于LNCaWHIF-1a／NSP，而且瘤体体积也远大于LNCaP／HIF-1cJNSP的成瘤体积[（1008±230）mm^3与（288±145）mm3，P〈0．01]。结论CSC是人前列腺癌中EMT现象发生的主要细胞载体。
Objective To test the hypothesis that epithelial-mesenchymal transition （EMT） of prostate cancer is most likely to occur in cancer stem cells （CSC）. Methods The isolation of CSC from LNCaP cell line was performed by flow cytometry based on side-population （SP） phenotype. After SP sorting, LNCaP/SP and LNCaP/NSP were used for further transfection of hypoxia-inducible factor-lot （HIF-lct）. Subsequently, EMT-associated proteins were detected by Western blotting. And the assays of Transwel] and methyl thiazolyl tetrazolium （MTT） were used to compare invasive and proliferative potency between LNCaP/ SP and LNCaP/NSP after HIF-la induction. Eventually, xenograft experiments were performed with LNCaP/ H1F-IcrJSP and LNCaP/HIF-la/NSP cells for further analysis of in vivo tumorigenesis and distant metastasis. Results Through HIF-loL-induced EMT, LNCaP/HIF-la/SP exhibited such remarkable EMT characteristics as a positive expression of epithelial markers （E-eadherin and CKI8 ） and a negative expression of mesenchymal markers （vimentin, N-cadherin, fibronectin, cathepsin D,MMP-2 and uPAR）. And LNCaP/HIF-la/NSP underwent partial EMT with an abnormal expression of some mesenchymalproteins （vimentin and cathepsin D） and loss of epithelial protein （CK18） despite reservation of another important epithelial marker （E-cadherin）. Further Transwell and MT＇F assays indicated that LNCaP/I-IIF-la/ SP exhibited stronger in vitro invasive and proliferative potency than LNCaP/HIF-1aNSP cells. In animal models, the volume of subcutaneous tumor by LNCaP/HIF-la/SP cells was much greater than that by LNCaP/ HIF-la/NSP counterparts （ （1008±230） vs （288 ±145） mm3, P 〈0. 01 ）. Moreover, LNCaP/HIF-1a/SP cells also had a significantly higher rate of subcutaneous tumor incidence （80% vs 53%, P 〈0. 05） and bone metastasis （40% vs 0, P 〈 0. O1 ） as compared with LNCaP/HIF-1a/NSP counterparts. Conclusion As the main target cells of prostatic EMT, CSCs may develop a more malignant phe
出处
《中华医学杂志》
CAS
CSCD
北大核心
2013年第4期256-260,共5页
National Medical Journal of China
基金
国家自然科学基金（30700968）
关键词
前列腺肿瘤
缺氧诱导因子1
A亚基
肿瘤干细胞
上皮细胞间质转化态
Prostatic neoplasms
Hypoxia-inducible factor 1, alpha subunit
Tumor stem cells
Epithelial-mesenchymal transition
