The Effect of Human Telomerase Reverse Transcriptase Repression on the Increasing Cell Viability and Alterations of Cell Cycle in Gastric Cancer Cell Line

Sogand Vahidi, Saba Sorayayi, Mohammad Mohammadzadeh, S Saied Hosseini-Asl

Abstract


Background:
Telomerase is a ribonucleoproteins enzyme responsible for the maintenance of telomere length. Human telomerase reverse transcriptase (hTERT) is a major component of the catalytic subunit of telomerase  enzymes and is expressed in cells that have telomerase activity but is not expressed in normal somatic cells. Based on the specific expression of hTERT in most cancer cells, it can be considered as a factor in the distinction between cancer cells and normal cells. It seems that inhibiting the expression of hTERT has been presented as a therapeutic approach in inhibiting the activity of telomerase. One of the special tools for inhibiting genes is the use of small interfering RNA (siRNAs). The purpose of this study was to investigate the effect of hTERT gene on the cell viability and cell cycle in gastric cancer cells.


Materials and Methods:
In this study, human cancer cells, adenocarcinoma gastric cell line (AGS) were cultured in RPMI 1640 medium (Roswell Park Memorial Institute) containing 10% FBS (Fetal Bovine Serum) and 1% penicillin/streptomycin antibiotics. The suppression of the hTERT gene was accomplished by FlexiTube siRNA. The repression effect of hTERT gene was investigated on cell viability by MTT assay [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] with ELISA (Enzyme Linked Immunosorbent Assay) reader at 570 nm, and cell cycle performed by flow cytometry and DAPI (4', 6-diamidino-2-phenylindole) staining.

Results:
The effect of hTERT siRNA on the cell viability by MTT assay showed time dependent cell viability of AGS cell line upon treatment and increasing the exposure time to 48 hours for that concentration decreased AGS cell (p = 0.02). Analysis of flow cytometry also showed increased number of cells in G1 phase and decreased the number of cells in S phase, and induced apoptosis via decreasing the level of hTERT expression.

Conclusion:
The significant downregulation in hTERT mRNA after 48 hours of hTERT siRNA treatment inhibited the cell viability of AGS cells and cell cycle arrest.


Keywords


Cancer cell line, Gastric cancer, hTERT, Cell viability, Cell cycle

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