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shRNA inhibits the expression of chicken telomerase reverse transcriptase in MDCC-MSB1 cells
N Jiang1, X B Zheng1, Z Y Zhao1
1China-Japan Union Hospital, Jilin University, Changchun, Jilin, China.
Abstract:
Here, we investigated the effects of blocking chicken telomerase reverse transcriptase (chTERT) in MDCC-MSB1 cells, using small-hairpin RNAs (shRNAs) to interfere with gene expression. shRNAs specific to chTERT mRNA were designed, cloned into DNA plasmid vectors, and transfected into MDCC-MSB1 cells. The transfected chTERT RNAs were expressed by the RNA polymerase machinery of the MDCC-MSB1 cells. mRNA expression in transfected MDCC-MSB1 cells was detected using real-time PCR. After transfection, telomerase activity was monitored via a modified telomeric repeat amplification protocol assay, and cell cycle analysis was performed using a flow cytometer. At 72 h after transfection, chTERT expression was considerably reduced in cells transfected with shRNA; the highest inhibition rate was 89%. Compared with the control group, telomerase activity was significantly reduced and the cells failed to progress to S phase. shRNA effectively reduced telomerase activity and prohibited the transition of MDCC-MSB1 cells from G2/M to S phase.
Insights
Blocking chicken telomerase reverse transcriptase (chTERT) with shRNAs significantly reduced telomerase activity in MDCC-MSB1 cells. This inhibition prevented cells from progressing from the G2/M phase to the S phase of the cell cycle.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Telomerase is crucial for maintaining telomere length and cellular proliferation.
- Chicken telomerase reverse transcriptase (chTERT) is the catalytic subunit of telomerase in chickens.
- Understanding chTERT function is important for research into cell proliferation and cancer.
Purpose of the Study:
- To investigate the effects of inhibiting chTERT expression in MDCC-MSB1 cells.
- To determine the impact of chTERT knockdown on telomerase activity and cell cycle progression.
Main Methods:
- Small-hairpin RNAs (shRNAs) were designed to target chTERT mRNA.
- shRNAs were cloned into DNA plasmid vectors and transfected into MDCC-MSB1 cells.
- chTERT expression was quantified using real-time PCR, telomerase activity was measured using a TRAP assay, and cell cycle analysis was performed by flow cytometry.
Main Results:
- Transfection with chTERT-specific shRNAs led to a significant reduction in chTERT expression, with inhibition rates up to 89%.
- Telomerase activity was substantially decreased in cells treated with shRNA compared to control cells.
- MDCC-MSB1 cells transfected with shRNA failed to progress from the G2/M phase to the S phase of the cell cycle.
Conclusions:
- shRNA-mediated knockdown of chTERT effectively reduces telomerase activity in MDCC-MSB1 cells.
- Inhibition of chTERT disrupts normal cell cycle progression, specifically blocking the G2/M to S phase transition.
- Targeting chTERT offers a potential strategy for controlling cell proliferation in relevant biological contexts.
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