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′ (HSP70B′) promoter and to examine the vector’s activities in RNAi induction and proliferation and apoptosis regulation 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 HSP70B′ 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 transfected 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 [63.45±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 suppress the proliferation and induce the apoptosis of MCF-7 cells under the condition of heat shock.

国家自然科学基金资助项目（No. 381273451）