Related Experiment Video
Updated: Jan 3, 2026
In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing
Published on: May 5, 2023
Targeted Inhibitory Effect of Nasopharyngeal Carcinoma Cells by Hre2.Grp78 Chimeric Promoter Regulating Fusion Gene
Jin-Yun Li1, Wen-Xiao Huang1, Jie Chen1
1Xiangya Hospital, Central South University, Changsha, China.
Objective:
To construct plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3 and elaborate the effects of overexpressed TK/VP3 on nasopharyngeal carcinoma cells.
Methods:
Four plasmids were constructed, including pcDNA3.1-CMV-TK/VP3, pcDNA3.1-Hre2.TK/VP3, pcDNA3.1-Grp78.TK/VP3, and pcDNA3.1-Hre2.Grp78.TK/VP3. The human nasopharyngeal carcinoma cell line HNE1 cells were transfected with the 4 plasmids, respectively. Cell viabilities were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was conducted using flow cytometry analysis. The expression of TK, VP3, Grp78, and hypoxia-inducible factor 1α and apoptosis-related proteins was determined by real-time quantitative polymerase chain reaction and Western blotting.
Results:
The recombinant plasmids that could steadily overexpress TK and VP3 were successfully constructed. Expression of TK and VP3 in cells transfected with pcDNA3.1-Hre2.TK/VP3 and pcDNA3.1-Grp78.TK/VP3 was significantly higher than pcDNA3.1-CMV-TK/VP3, and expression in cells transfected with pcDNA3.1-Hre2.Grp78.TK/VP3 was the highest. Under glucose deprivation or hypoxia condition, Grp78 or hypoxia-inducible factor 1α was overexpressed so that expression of TK and VP3 was significantly upregulated, which could further inhibit cell proliferation and enhance cell apoptosis.
Conclusion:
We successfully constructed 4 plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3, which could significantly inhibit the proliferation as well as enhance the apoptosis of nasopharyngeal carcinoma cells under glucose deprivation or hypoxia condition.
Insights
Engineered plasmids overexpressing TK/VP3 fusion genes significantly inhibit nasopharyngeal carcinoma cell proliferation and enhance apoptosis, particularly under hypoxia or glucose deprivation. This offers a novel therapeutic strategy for cancer treatment.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Regulation
Background:
- Nasopharyngeal carcinoma (NPC) remains a significant health challenge.
- Targeted gene therapy offers potential for novel cancer treatments.
- Understanding gene regulation under stress conditions is crucial for therapeutic development.
Purpose of the Study:
- To construct and characterize novel plasmids for overexpressing the TK/VP3 fusion gene in NPC cells.
- To investigate the effects of the Hre2.Grp78 chimeric promoter on TK/VP3 expression.
- To evaluate the impact of TK/VP3 overexpression on NPC cell proliferation and apoptosis.
Main Methods:
- Construction of four distinct plasmids, including controls and those with the Hre2.Grp78 promoter.
- Transfection of human NPC cell line (HNE1) with constructed plasmids.
- Assessment of cell viability (MTT assay) and apoptosis (flow cytometry).
- Quantification of gene and protein expression (RT-qPCR, Western blotting).
Main Results:
- Successful construction of recombinant plasmids enabling stable TK/VP3 overexpression.
- Hre2.Grp78 promoter significantly enhanced TK/VP3 expression compared to CMV promoter.
- Overexpression of TK/VP3 inhibited NPC cell proliferation and induced apoptosis.
- Expression of TK/VP3 was further upregulated under glucose deprivation or hypoxia.
Conclusions:
- The Hre2.Grp78 chimeric promoter effectively drives TK/VP3 fusion gene expression in NPC cells.
- Overexpression of TK/VP3 demonstrates potent anti-cancer effects by inhibiting proliferation and promoting apoptosis.
- These findings support the potential of TK/VP3 as a therapeutic agent for nasopharyngeal carcinoma, especially under stress conditions.
More Related Videos
11:08Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells
Published on: February 14, 2025
08:29Engineering Chimeric Antigen Receptor-Natural Killer Cells Targeting Fungal Infections Using the Non-viral Sleeping Beauty Transposon System
Published on: October 4, 2024
