摘要
目的明确β-连环蛋白（catenin）信号通路是否参与缺氧诱导因子（HIF）-1α诱导人前列腺癌细胞发生上皮细胞间质转化态（EMT）转化过程。方法应用Western印迹法检测HIF-1α、Glut-1和VEGF蛋白表达，确认2种新构建前列腺癌细胞株（LNCaP／HIF1α和PC-3／HIF1α）的稳定性；然后应用Western印迹法检测EMT指标蛋白（E—cadherin、CK18、Vimentin、N-cadherin及Fibronetin）的表达，对5种人前列腺癌细胞株（LNCaP、LNCaP／HIF1α、PC-3、PC-3／HIF1α和IA8）的EMT特性进行鉴定；进一步应用Transwell和MTT技术检测5种细胞株的体外侵袭和增殖潜能；最后，用RT-PCR和Western印迹法检测5种EMT特性不同的细胞株中β—catenin、tGSK-3β和pGSK-3β的表达，总结分析该信号通路活性与细胞EMT特性的关联。结果（1）LNCaP／HIF1α和PC-3／HIF1α中出现明显的HIF-1α蛋白条带，同时Glut-1和VEGF表达呈强阳性；（2）PC-3、LNCaP和PC-3／HIF1α是EMT阴性细胞，而LNCaP／HIF1α和IA8是EMT阳性细胞；（3）PC-3／HIF1α和LNCaP／HIF1α、IA8体现出了较PC-3和LNCaP更为强大的体外侵袭和增殖潜能；（4）与LNCaP和PC-3相比，PC-3／HIF1α和LNCaP/HIF1α、IA8中tGSK-3β和pGSK-3β的蛋白表达相对减少，但P—GSK3β／t-GSK3β比值相应较高，同时，β—catenin蛋白表达在LNCaP／HIF1α和IA8中相对较高，PC-3／HIF1α却表达较低；基因检测结果与前述蛋白表达规律基本一致，但是与β-catenin蛋白在PC-3／HIF1α中低表达不吻合的是，PC-3／HIF1α中β—cateninmRNA水平与其在LNCaP／HIF1α和IA8中一样呈现出强表达特点。结论β-catenin信号通路的活性状态与细胞EMT特性及其体外侵袭和增殖潜能有密切关系，该信号通路可能是介导HIF-1α诱导人前列腺癌细胞EMT过程的重要“桥梁”。
Objective Epithelial-mesenchymal transition （EMT） is an important process in tumor development. Several studies suggest that the β-catenin signal pathway may p1αy an important role in EMT. However, there is no direct evidence showing that this pathway actually determines the EMT induced by exogenous signal. Our previous study has successfully proved that over-expression of HIF-1α could induce EMT in LNCaP cells, but not in PC-3. So the present study was intended to indicate that the signal of HIF- 1α for inducing prostate cancer cell to undergo EMT might pass through the β-catenin pathway. Methods Firstly, we analyzed the expression of HIF-1α and its target proteins in LNCaP/HIF1α and PC-3/HIF1α by Western b1α. And then EMT-associated proteins were detected by Western b1α. Furthermore the potency of invasiveness and proliferation of several cell lines were evaluated by transwell and MTT assay. Lastly the expressions of β-catenin and GSK-3β in these cells were analyzed by Western b1α and RT-PCR. Results HIF-1α, Glut-1 and VEGF were highly expressed in LNCaP/HIF1α and PC-3/HIF1α. And PC-3, LNCaP and PC-3/HIF1α were EMT-negative cell lines whereas LNCaP/HIF1α and IA8 had undergone EMT process. LNCaP/HIF1α, IA8 and PC-3/HIF1α exhibited a much stronger potency of invasiveness and proliferation than those of PC-3 and LNCaP. The protein levels of total GSK-3β and phospho-GSK-3β in LNCaP/HIF1α, IA8 and PC-3/HIF1α cells significantly decreased; however, the relative ratios of p-GSK3β/t-GSK3β increased. Consistently, β-catenin protein expression increased in LNCaP/HIF1α and IA8 cells, but not in PC-3/HIF1α; RT-PCR confirmed these results, except for an enhanced transcription activity of β-catenin mRNA in PC-3/HIF1α. Conclusion The activation of the β-catenin signaling pathway correlates with the characteristic of EMT and potency of invasiveness and proliferation. And it may be one critical factor directly controlling the process of EMT induced by HIF-1α in prostate cancer.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2010年第16期1131-1136,共6页
National Medical Journal of China
基金
国家自然科学基金（30700968）
