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Archive > Volume 21 Issue 2 > 2014,21(2):130-135. DOI:10.3872/j.issn.1007-385X.2014.02.003 Prev Next
Prompt Report
Construction of an HSP 70B′ promoter-driven heat-inducible vectors pHSP-shTERT and its anti-proliferative effect in breast cancer MCF-7 cells
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Abstract:
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.
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Project Supported:
Project supported by the National Natural Science Foundation of China （No. 381273451）
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