Background & Objective: RNA interference (RNAi) is a new technology in gene study. The mechanism of RNAi is that double-stranded RNA (dsRNA) can band target mRNA and decompose it. This study was to assess possibility and specificity of dsRNA on suppressing human telomerase reverse transcriptase (hTERT) in lung carcinoma cells, investigate its effect on cell proliferation to confirm whether it has unspecific killing activity on mammalian cells, and explore its application in lung cancer research and treatment.
Methods: Sequences of 2 exons and 1 intron of hTERT gene were amplified by reverse transcription-polymerase chain reaction (RT-PCR) or PCR. The sense and antisense cDNA sequences were connected in a tandem manner, and the whole fragment was inserted into pCI-neo mammalian expression vector to construct the dsRNA expression vector, and then transfected into lung carcinoma cell line A549. The expression of hTERT was detected by RT-PCR and Western blot. Telomerase activity was measured by telomerase repeat amplification protocol (TRAP). Cell morphology was observed, and cell proliferation was assessed under invert microscope.
Results: After transfection of 2 exon fragments of hTERT dsRNA, mRNA and protein expression of hTERT and telomerase activity in A549 cells were suppressed, cell proliferation was markedly inhibited. Meanwhile, dsRNA didn't show unspecific toxic activity on A549 cells.
Conclusions: hTERT dsRNA can specifically silent hTERT gene, inhibit telomerase activity and proliferation of A549 cells. hTERT dsRNA might be a potential method of gene therapy for lung cancer.
Download full-text PDF |
Source |
|---|
Publication Analysis
Top Keywords
Similar Publications
Strengthening of the barrier function in human telomerase reverse transcription (hTERT) immortalized corneal and conjunctival epithelium by double-stranded RNA.
Exp Eye Res
February 2023
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Article Synopsis
- The study investigates how poly(I:C), a compound that simulates viral infection, affects the barrier function of two human cell lines: HCLE (human corneal-limbal epithelial) and HCjE (human conjunctival-epithelial).
- Researchers used techniques like Western blotting and TEER measurements to analyze the expression of tight junction (TJ) proteins and overall barrier function before and after poly(I:C) stimulation.
- Results showed that both cell lines exhibited increased levels of certain claudin proteins and enhanced barrier function after treatment, suggesting this response may help protect against viral infections.
Molecular Recognition and Imaging of Human Telomeric G-Quadruplex DNA in Live Cells: A Systematic Advancement of Thiazole Orange Scaffold To Enhance Binding Specificity and Inhibition of Gene Expression.
J Med Chem
February 2021
School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, P. R. China.
A series of fluorescent ligands, which were systematically constructed from thiazole orange scaffold, was investigated for their interactions with G-quadruplex structures and antitumor activity. Among the ligands, compound was identified to exhibit excellent specificity toward telomere G4-DNA over other nucleic acids. The affinity of -Htg24 was almost 5 times higher than that of double-stranded DNA and promoter G4-DNA.
View Article and Find Full Text PDFEnhancements in the utilization of antigene oligonucleotides in the nucleus by booster oligonucleotides.
Chem Commun (Camb)
August 2020
Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan.
We recently found the translocation of double-stranded DNA into the nucleus. We herein describe the concept of novel booster oligodeoxynucleotides including 2'-deoxy uridine, which release antigene oligonucleotides in the nucleus by enzymatic digestion. This system exhibited stronger hTERT mRNA expression inhibitory activity than single-stranded antigene oligonucleotides.
View Article and Find Full Text PDFhTERT gene knockdown enhances response to radio- and chemotherapy in head and neck cancer cell lines through a DNA damage pathway modification.
Sci Rep
April 2018
Department of Head and Neck Surgery, Poznan University of Medical Sciences, The Greater Poland Cancer Centre, Garbary 15 Str., 61-866, Poznan, Poland.
The aim of the study was to analyze the effect of hTERT gene knockdown in HNSCC cells by using novel in vitro models of head and neck cancer (HNSCC), as well as improving its personalized therapy. To obtain the most efficient knockdown siRNA, shRNA-bearing lentiviral vectors were used. The efficiency of hTERT silencing was verified with qPCR, Western blot, and immunofluorescence staining.
View Article and Find Full Text PDFPost-transcriptional regulation of BRCA1 through its coding sequence by the miR-15/107 group of miRNAs.
Front Genet
August 2015
Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia PA, USA.
MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by degrading their RNA targets or by repressing the translation of messenger RNAs (mRNAs). Initially thought to primarily target the 3' untranslated region (3'UTR) of mRNAs, miRNAs have since been shown to also target the 5'UTR and coding sequence (CDS). In this work, we focus on the post-transcriptional regulation of the BRCA1 gene, a major tumor suppressor and regulator of double-stranded break DNA repair and show that its mRNA is targeted by many members of the miR-15/107 group at a site located within the CDS.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!