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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 construct the recombinant plasmid pGL3-TFDP3-promoter containing TFDP3 promoter and luciferase reporter gene and to observe the regulatory effect of E2F1 on the transcription and expression of TFDP3 and the effect of TFDP3 on E2F1-induced tumor cell apoptosis. Methods: The genomic DNA of human prostate cancer PC3 cell line was used as template to amplify the TFDP3 promoter sequence and cloned into luciferase reporter gene vector. PC3 cells were transfected with E2F1 expression vector pCMV-E2F1-HA transiently or separately Luciferase activity to observe the effect of E2F1 on the TFDP3 promoter. Western blotting was used to detect the effect of pCMV-E2F1-HA transfection on the expression of TFDP3 in PC3 cells. Flow cytometry was used to detect the interaction between TFDP3 and E2F1 on the apoptosis of prostate cancer cells Impact. Results: After co-transfecting PC3 cells with E2F1 expression vector pCMV-E2F1-HA, the TFGL3-TFDP3-promoter was successfully constructed and the luciferase activity induced by TFDP3 promoter was significantly lower than that of pGL3-TFDP3-promoter Significantly increased [(1.14 ± 0.06) vs (0.61 ± 0.05), P <0.05]. TFDP3 protein expression of PC3 cells transfected with pCMV-E2F1-HA was 2.7-fold more than that of untransfected cells [(0.24 ± 0.03) vs (0.09 ± 0.02) P <0.05]. The apoptosis rate of PC3 cells after transfection with pCMV-E2F1-HA was significantly higher than that of untransfected cells [(7.10 ± 0.003)% vs (2.66 ± 0.001)%, P <0.05] Compared with pCMV-E2F1-HA group, the apoptotic rate of TDPDP3-co-transfected cells was significantly decreased (4.92 ± 0.002)% vs (7.10 ± 0.003)%, P <0.05. Conclusion: E2F1 can enhance the activity of TFDP3 promoter and increase the expression of TFDP3 protein, which may inhibit E2F1-induced apoptosis of prostate cancer cells by this mechanism.