Luo Yong, He Da-Lin, Ning Liang, Shen Shu-Lin, Li Lei, Li Xiang, Zhau Haiyen E, Chung Leland W K
Department of Urology, First Hospital of Xi'an Jiaotong University, Xi'an Jiaotong University, Ministry of Education, Xi'an, People's Republic of China.
BJU Int. 2006 Dec;98(6):1315-9. doi: 10.1111/j.1464-410X.2006.06480.x. Epub 2006 Sep 6.
To evaluate the effect of hypoxia-inducible factor-1alpha (HIF-1alpha) over-expression on the invasion-associated proteins in human prostate cancer cells, as HIF-1alpha is a transcriptional factor that could activate genes involved in the response to hypoxia, but might also enhance the invasive potency of prostate cancer cells.
Prostate cancer (LNCaP) cells were transfected with recombinant plasmid pcDNA3.1(-)/HIF-1alpha and pcDNA3.1(-) control vector using a commercial system, designated as LNCaP/HIF-1alpha and LNCaP/pcDNA3.1, respectively. The positive clones were selected with G418 and confirmed by Western blot and indirect immunofluorescence labelling. A polycarbonate filter, coated with a matrix gel, was used to analyse the invasive potency. The expression of E-cadherin, vimentin, cathepsin D, matrix metalloproteinase (MMP2) and urokinase plasminogen activator receptor (uPAR) was detected by Western blot.
The expression level of HIF-1alpha in LNCaP/HIF-1alpha was distinctly higher than that in LNCaP/pcDNA3.1 and LNCaP. Many more cells of LNCaP/HIF-1alpha penetrated through the polycarbonate filter than those of LNCaP/pcDNA3.1 and LNCaP. Compared with the LNCaP/pcDNA3.1 and LNCaP cells, the expression of vimentin, cathepsin D, MMP-2 and uPAR were up-regulated in LNCaP/HIF-1alpha, whereas the expression of E-cadherin was down-regulated.
These results show that over-expression of HIF-1alpha directly stimulates the invasive potency of human prostate carcinoma LNCaP cells through the matrix gel. The expression of E-cadherin, vimentin, cathepsin D, MMP-2 and uPAR, which are proteins with established roles in the pathophysiology of invasion, could be regulated by HIF-1alpha in human prostate cancer cells.
评估缺氧诱导因子-1α（HIF-1α）过表达对人前列腺癌细胞中侵袭相关蛋白的影响，因为HIF-1α是一种转录因子，可激活参与缺氧反应的基因，但也可能增强前列腺癌细胞的侵袭能力。
使用商业系统将重组质粒pcDNA3.1(-)/HIF-1α和pcDNA3.1(-)对照载体转染前列腺癌（LNCaP）细胞，分别命名为LNCaP/HIF-1α和LNCaP/pcDNA3.1。用G418筛选阳性克隆，并通过蛋白质免疫印迹法和间接免疫荧光标记进行确认。使用涂有基质胶的聚碳酸酯滤膜分析侵袭能力。通过蛋白质免疫印迹法检测E-钙黏蛋白、波形蛋白、组织蛋白酶D、基质金属蛋白酶（MMP2）和尿激酶型纤溶酶原激活剂受体（uPAR）的表达。
LNCaP/HIF-1α中HIF-1α的表达水平明显高于LNCaP/pcDNA3.1和LNCaP。与LNCaP/pcDNA3.1和LNCaP相比，LNCaP/HIF-1α穿过聚碳酸酯滤膜的细胞更多。与LNCaP/pcDNA3.1和LNCaP细胞相比，LNCaP/HIF-1α中波形蛋白、组织蛋白酶D、MMP-2和uPAR的表达上调，而E-钙黏蛋白的表达下调。
这些结果表明，HIF-1α的过表达通过基质胶直接刺激人前列腺癌LNCaP细胞的侵袭能力。E-钙黏蛋白、波形蛋白、组织蛋白酶D、MMP-2和uPAR这些在侵袭病理生理学中具有既定作用的蛋白质的表达，可在人前列腺癌细胞中受HIF-1α调节。
