E2F1对TFDP3表达的调控及其对前列腺癌PC3细胞凋亡的影响

Role for E2F1 in the regulation of transcription factor dimerization partner-3 expression and apoptosis in prostate cancer PC3 cells

目的:构建含TFDP3基因启动子和荧光素酶报告基因的重组载体pGL3-TFDP3-promoter,观察E2F1对TFDP3转录及表达的调控作用以及TFDP3对E2F1诱导肿瘤细胞凋亡的影响。方法:以人前列腺癌PC3细胞系基因组DNA为模板,PCR扩增TFDP3启动子序列并克隆入荧光素酶报告基因载体,与E2F1表达载体pCMV-E2F1-HA瞬时单独或共同转染PC3细胞,测定荧光素酶活性以观察E2F1对TFDP3启动子的调控作用,Western blotting检测pCMV-E2F1-HA转染对PC3细胞内TFDP3表达的影响,流式细胞术检测TFDP3与E2F1相互作用对前列腺癌细胞凋亡的影响。结果:成功构建TFDP3基因启动子重组质粒pGL3-TFDP3-promoter,与E2F1表达载体pCMV-E2F1-HA共转染PC3细胞后,TFDP3启动子诱导的荧光素酶活性较单独转染pGL3-TFDP3-promoter显著升高[(1.14±0.06)vs(0.61±0.05),P<0.05]。转染pCMV-E2F1-HA的PC3细胞的TFDP3蛋白表达是未转染细胞的2.7倍[(0.24±0.03)vs(0.09±0.02),P<0.05]。pCMV-E2F1-HA转染后PC3细胞凋亡率较未转染组显著上升[(7.10±0.003)%vs(2.66±0.001)%,P<0.05],而pCMV-E2F1-HA与pcDNA3.1-TFDP3共转染后细胞凋亡率较pCMV-E2F1-HA组显著下降[(4.92±0.002)%vs(7.10±0.003)%,P<0.05]。结论:E2F1可增强TFDP3启动子的活性,增加TFDP3蛋白的表达,其可能通过此机制抑制E2F1诱导的前列腺癌细胞凋亡。

Objective： To evaluate the regulatory effects of E2F1 on transcription factor dimerization partner-3 （TFDP3） expression and apoptosis in prostate cancer cells in vitro. Methods： A luciferase reporter construct driven by the human TFDP3 gene promoter, pGL3-TFDP3-promoter, was made through PCR and subcloning using the total DNA ex- tracted from human prostate cancer PC3 cells. PC3 cells were transfected with pGL3-TFDP3-promoter and pCMV-E2F1- HA E2F1 expression vector, either alone or together. TFDP3 promoter activity and TFDP3 protein content in the transfec- tants were determined by dual luciferase assays and Western blotting analysis, respectively, 48 h after transfection, while apoptosis was analyzed by flow cytometry 24 h after transfection. Results： The lueiferase activity was significantly higher in PC3 cells eo-transfected with pGI3-TFDP3-promoter and pCMV-E2F1-HA than PC3 cells transfected with pGL3- TFDP3-promoter alone （ 1.14 vs 0. 61, P 〈 0.05）. TFDP3 protein content in PC3 cells transfected with pCMV-E2F1-HA was 2.7 times higher than that in non-transfected cells （ [ 0.24 ± 0. 03 ] vs [ 0. 089 ± 0.02 ], P 〈 0.05 ）. The proportion of apoptotic cells PC3 cells transfected with pCMV-E2F1-HA （7.1 ± 0. 003 ）% was significantly higher than that both in non-transfected PC3 cells （ [ 2.66 ± 0. 001 ] % P 〈 0.05 ） and in PC3 cells co-transfected with pcDNA3-TFDP3-promoter and pCMV-E2F1-HA （ [ 4.92 ± 0. 002 ] % vs [ 7.1 ± 0. 003 ] %, P 〈 0.05 ）. Conclusion： E2F1 may enhance the TFDP3 promoter activity and upregulate TFDP3 expression in prostate cancer cells. This finding suggests that E2F1 and TFDP3may play a role in the survival/apoptosis in prostate cancer cells.