热激蛋白70B'启动子调控的热诱导型载体pHSP-shTERT的构建及其对乳腺癌MCF-7细胞的抑制效应

Construction of an HSP 70B' promoter-driven heat-inducible vectors pHSPshTERT and its anti-proliferative effect in breast cancer MCF-7 cells

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 novel heat-inducible RNAi vector under the control of the heat shock protein 70B＇ （HSPTOB＇） promoter and to examine the vector＇ s activities in RNAi induction and proliferation and apoptosis regu- lation in MCF-7 cells under the condition of heat shock. Methods： An HSP70B＇ promoter-driven heat-inducible RNAi vectors against telomerase reverse transcriptase （TERT）, pHSP-shTERT, was constructed through PCR-amplification of the HSPTOB＇ promoter and subsequent subcloning. MCF cells were optimally transfected with this vector with GFP as an indicator and flow cytometric analysis as a confirmatory assay. Untransfected MCF-7 cells were used as a control. TERT mRNA and protein levels were analyzed by RT-PCR and Western blotting, respectively. Cell proliferation and apotosis were determined by MTT assay and flow cytometry, respectively. Results： As compared with control cells, cells transfect- ed with newly constructed vector pHSP-shTERT had significantly lower levels of TERT mRNA （ [ 12.24 ± 1.96 ] % vs [ 80.18 ± 2.28 ] % ; t = - 286.5, P = 0. 000 012） and protein （ [ 1.64 ± 0.42 ] % vs [ 53.45 ± 3.12 ] % ; t = - 31.37,P = 0. 001 ）, significantly decreased cell viability （ [ 58.93 -± 2.95 ] % vs [ 91.22 ± 4.16 ] % ; t = 15. 747, P = 0. 004 ） and significantly higer apoptosis rate （ [40.97 ±4.80] % vs [8.33 ± 1.14] ; t = - 11. 672, P =0. 007）. Conclusions： The novel heat inducible vector pHSP-shTERT constructed against TERT was effective to silence the TERT gene and sup- press the proliferation and induce the apoptosis of MCF-7 cells under the condition of heat shock.