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Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy
Published on: April 3, 2018
Antitumor effects of targeting hTERT lentivirus-mediated RNA interference against KB cell lines
Dan Chen1, Hongzhang Huang, Chaobin Pan
1Department of Oral & Maxillofacial Surgery, Guanghua College of Stomatology, Sun Yat-Sen University, Guangzhou, Guangdong Province, 510055, PR China.
Abstract:
Squamous cell carcinomas are the leading frequent malignant tumors in the oral and maxillofacial region. Currently available treatment options are of limited efficacy, and there is an urgent need for development of alternative therapies. RNA interference (RNAi) is a sequence-specific RNA degradation process. In this study, we screened and identified an in vitro-transcribed 21-bp shRNA targeting human telomerase reverse transcriptase (hTERT) from three candidates and generated a lentivirus vector. Subsequent experiments indicated that this lentiviral transgenic system could effectively transfer into target KB cells, above 80% gene transfer efficiency at MOI of 2.5, and significantly and specifically inhibited hTERT expression both in mRNA (73.42%) and protein (74.67-82.91%) levels. To further evaluate the role of hTERT-targeted RNAi, we found that hTERT inhibition consequently induced suppression of cyclin D1 (54.67%), upregulation of caspase-3 (100.10%), and caspase-9 (42.67%) of KB cells. Therefore, the apoptosis rates of KB cells were increased by 206.33%. In conclusion, these data indicated the potential of lentivirus vectors in cancer gene therapy, especially after development of more efficient vector production methods, and higher virus titers demonstrated that targeting hTERT RNAi may result in telomere uncapping, which triggers cell cycle arrest and apoptosis signal and leads to tumor suppression.
Insights
RNA interference targeting human telomerase reverse transcriptase (hTERT) effectively suppressed oral cancer cells. This lentiviral gene therapy approach shows promise for developing novel cancer treatments by inducing apoptosis.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- Oral squamous cell carcinomas are prevalent and current treatments have limited efficacy.
- There is a critical need for innovative therapeutic strategies.
- RNA interference (RNAi) offers a sequence-specific approach to gene silencing.
Purpose of the Study:
- To develop and evaluate an RNA interference-based therapy targeting human telomerase reverse transcriptase (hTERT) for oral cancer.
- To assess the efficacy of a lentiviral vector system for delivering hTERT-targeted shRNA into cancer cells.
Main Methods:
- Screening and identification of a 21-bp shRNA targeting hTERT.
- Generation of a lentivirus vector for shRNA delivery.
- Transduction of KB oral cancer cells and assessment of gene transfer efficiency, hTERT expression inhibition, and downstream effects on cell cycle and apoptosis markers.
Main Results:
- The lentiviral system achieved >80% gene transfer efficiency in KB cells.
- Significant inhibition of hTERT expression at both mRNA (73.42%) and protein (74.67-82.91%) levels was observed.
- hTERT inhibition led to suppressed cyclin D1, upregulated caspases-3 and -9, and a 206.33% increase in KB cell apoptosis.
Conclusions:
- Lentivirus-mediated delivery of hTERT-targeted RNAi is a potent strategy for oral cancer gene therapy.
- Targeting hTERT induces cell cycle arrest and apoptosis, leading to tumor suppression.
- Further development of efficient vector production and higher virus titers could enhance therapeutic potential.
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