缺氧诱导因子1α诱导人前列腺癌细胞上皮间质化改变的研究

目的:研究人前列腺癌细胞在缺氧诱导因子1α(HIF-1α)诱导下能否发生上皮细胞间质转化态(EMT)改变,进而致侵袭能力增强,并初步分析其分子机制。方法:应用RT-PCR技术检测LNCaP细胞及其亚细胞系C4、C4-2、C4-2B这4种EMT阴性的人前列腺癌细胞中波形蛋白(vimentin)mRNA表达情况,并凭此筛选出适合于进一步作转染诱导试验的细胞。用脂质体Lipofectamine2000包装重组真核表达载体pCDNA3.1(-)/HIF-1α和pCD-NA3.1(-)空质粒后,分别转染上步试验所挑选出的人前列腺癌细胞LNCaP,600μg/mLG418筛选抗性克隆。免疫荧光及Western印迹法鉴定HIF-1α表达,Western印迹法检测EMT标志蛋白——上皮型钙粘素(E-cadherin)和vimentin的表达,Transwell验证转染后LNCaP细胞侵袭能力的改变。结果:RT-PCR证实4种EMT阴性的细胞中,仅LNCaP表达有vimentin编码基因,适合作转染诱导试验。免疫荧光也观察到HIF-1α转染细胞胞质中荧光亮度较空质粒转染细胞和未转染细胞明显增强。Western印迹法证实HIF-1α转染细胞发生了EMT转化,其E-cad-herin表达缺失,而vimentin表达增加。同时,Transwell体外侵袭试验也发现,LNCaP/HIF1α细胞的体外侵袭能力显著高于LNCaP细胞和LNCaP/pCDNA3.1(-)细胞。结论:HIF-1α过表达可以通过调节两种EMT相关蛋白诱导人前列腺癌细胞LNCaP发生EMT改变并致其侵袭能力增强。

Over-expression of HIF-1α induces EMT of Human Prostate Cancer Cells

Objective： To determine whether human prostate cancer cell lines undergo epithelial-mesenchymal transition （EMT） and become more invasive when induced by HIF-1α, and to explore the underlying molecular mechanism. Methods： The cell line LNCaP, appropriate for the HIF1α induction test, was screened out from 4 different EMT-negative prostate cell lines through vimentin gene detection by RT-PCR. The recombinant plasmid pCDNA3.1 （ -）/HIF-1α was constructed and transfected into LNCaP with the Lipofectamine 2000 system. The control plasmid pCDNA3.1 （-） was transfected by the same method. The positive clone cells were selected by G418 and confirmed by Western blot and immunofluorescence staining. Then a Transwell polycarbonate filter, coated with 100ul Matrigel at 1：20 dilution in the serum-free medium, was used to analyze the invasive potency. The expression of E-cadberin and vimentin was detected by Western blot. Results： Among the 4 different EMT-negative cell lines, LNCaP was the only one that expressed the vimentin gene but not protein. The expression of HIF1α was obviously higher in LNCaP/HIF1α than in LNCaP/pCDNA3. 1 （-） and LNCaP. The number of the LNCaP/HIF1α cells that penetrated through the Transwell polycarbonate filter was significantly larger than that of the LNCaP and LNCaP/pCDNA3.1 （-） cells. Compared with the LNCaP/pCDNA3.1 （-） and LNCaP cells, the expression of vimentin was up-regulated, while that of E-eadherin down-regulated, in LNCaP/HIF1α. Conclusion： The over-expression of HIF-1α could induce EMT in the human prostate carcinoma cell line LNCaP and enhance its invasiveness through E-eadherin and vimentin regulation.