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目的:构建靶向端粒酶逆转录酶(telomerose reverse transcriptas,TERT)的热激蛋白(heat shock protein,HSP)70B’启动子调控的热诱导型RNAi表达载体,观察热诱导条件下其在人乳腺癌MCF-7细胞内的RNAi效应及其对MCF-7细胞增殖和凋亡的影响。方法:PCR扩增HSP70B’启动子序列,用其构建热诱导型靶向TERT的重组载体并转染MCF-7细胞,倒置荧光显微镜和流式细胞术检测GFP的表达,优化转染和热诱导条件;以未经热诱导的转染细胞为对照组,RT-PCR和Western blotting检测重组载体转染与热诱导对MCF-7细胞内GFP与TERT的mRNA和蛋白表达的影响;MTT实验和流式细胞术分别检测对MCF-7细胞增殖与凋亡的影响。结果:成功构建热诱导型重组载体pHSP-GFP、pHSP-shGFP和pHSP-shTERT;在最适热诱导条件(42℃、40 min)下,共转染质粒pHSP-shGFP和pCMV-GFP的MCF-7细胞中GFP表达量显著低于对照组(t=-48.35,P=0.000 43)。pHSP-shTERT转染组MCF-7细胞内TERT mRNA[(12.24±1.96)%vs(80.18±2.28)%;t=-286.5,P=0.000 012]和蛋白[(1.64±0.42)%vs(63.45±3.12)%;t=-31.37,P=0.001]的表达均显著下调,细胞存活率显著下降[(58.93±2.95)%vs(91.22±4.16)%;t=15.747,P=0.004],细胞凋亡率显著提高[(40.97±4.80)%vs(8.33±1.14)%;t=-11.672,P=0.007]。结论:靶向TERT的pHSP-shTERT载体可以在热诱导条件下实现MCF-7细胞内靶基因的沉默,从而抑制MCF-7细胞的生长、诱导该细胞的凋亡。
OBJECTIVE: To construct a heat-inducible RNAi expression vector targeting the heat shock protein (HSP) 70B promoter targeting telomerose reverse transcriptase (TERT) Effect of RNAi in Breast Cancer MCF-7 Cells on Proliferation and Apoptosis of MCF-7 Cells. Methods: The HSP70B promoter was amplified by PCR. The recombinant vector was constructed and transfected into MCF-7 cells. The expression of GFP was detected by inverted fluorescence microscope and flow cytometry. The transfection and heat-induced RT-PCR and Western blotting were used to detect the expression of GFP and TERT mRNA and protein in MCF-7 cells transfected with recombinant vector and heat-induced; MTT assay and flow cytometry Cytometry were detected on MCF-7 cells proliferation and apoptosis. Results: The heat-inducible recombinant vectors pHSP-shGFP and pHSP-shTERT were constructed successfully. The recombinant plasmids pHSP-shGFP and pCMV-GFP were co-transfected into MCF- 7 cells was significantly lower than the control group (t = -48.35, P = 0.00043). TERT mRNA in MCF-7 cells transfected with pHSP-shTERT group was significantly higher than that in control group [(12.24 ± 1.96)% vs (80.18 ± 2.28)%; t = -286.5, P = 0.000121 and protein [(1.64 ± 0.42)% vs ± 3.12)%; t = -31.37, P = 0.001] were significantly down-regulated and the cell viability was significantly decreased [(58.93 ± 2.95)% vs (91.22 ± 4.16)%; t = 15.747, P = 0.004] The apoptotic rate increased significantly ([(40.97 ± 4.80)% vs (8.33 ± 1.14)%; t = -11.672, P = 0.007]. CONCLUSION: The pHSP-shTERT vector targeting TERT can silence the target gene in MCF-7 cells under heat-induced conditions, thereby inhibiting the growth of MCF-7 cells and inducing the apoptosis of MCF-7 cells.