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Published on: March 30, 2019
Use of RNA interference to elucidate the effect of MYCN on cell cycle in neuroblastoma
Chan-Wook Woo1, Fei Tan, Hope Cassano
1Department of Pediatrics, College of Medicine, Korea University, Seoul 152-703, South Korea. wooc@korea.ac.kr
Background:
MYCN amplification marks poor prognosis in neuroblastoma (NB) tumors. In evaluating the mechanisms by which retinoic acid (RA) or nerve growth factor (NGF) decrease cell number in MYCN amplified NB cells, we have identified a number of proteins whose expression either decreases (E2F, CDC2, CDK6, cyclin dependent kinase activity) or increases (p27) in association with a decrease in MYCN expression. However, it was still unclear which were MYCN dependent effects or not.
Procedure:
This study aimed to determine which changes in cell cycle gene expression are modulated as a consequence of the decrease in MYCN. We silenced MYCN expression using siRNA targeted to the coding region of MYCN. Then, by using siRNA transient transfections, we analyzed the change of cell cycle related genes and cell cycle in MYCN amplified NB cell lines.
Results:
We demonstrate that expression of MYCN can be suppressed by almost 60% in MYCN amplified NB cell using siRNAs targeted to MYCN. Functionally, the decrease in MYCN leads to a decrease in cells in the S-phase of the cell cycle. Decreases in MYCN are associated with decreases in E2F1-2 and ID2 along with increases in p27 protein levels by post-transcriptional modification. Moreover, we find that a decrease in MYCN is accompanied by a decrease in cdk6 mRNA and protein expression.
Conclusions:
These results show that E2F and ID2 expression is associated with MYCN regulation and that cdk6 is a possible new transcriptional target of MYCN.
Insights
MYCN amplification in neuroblastoma is linked to poor prognosis. Silencing MYCN reduces S-phase cells and affects cell cycle genes like E2F, ID2, p27, and cdk6, revealing MYCN
Area of Science:
- Neuro-oncology
- Molecular biology
- Cell cycle regulation
Background:
- MYCN amplification is a key driver of poor prognosis in neuroblastoma (NB).
- Understanding how MYCN influences cell cycle progression is crucial for developing targeted therapies.
- Previous studies noted changes in cell cycle proteins with MYCN modulation, but MYCN dependency was unclear.
Purpose of the Study:
- To elucidate which cell cycle gene expression changes are directly modulated by MYCN.
- To investigate the functional consequences of MYCN suppression on cell cycle progression in NB cells.
Main Methods:
- Utilized small interfering RNA (siRNA) to specifically silence MYCN expression in MYCN-amplified NB cell lines.
- Analyzed changes in the expression of cell cycle-related genes and overall cell cycle distribution post-MYCN knockdown.
Main Results:
- Successfully suppressed MYCN expression by approximately 60% using MYCN-targeted siRNAs.
- Demonstrated that MYCN reduction leads to decreased S-phase cell population.
- Observed decreased E2F1-2 and ID2 expression, alongside increased p27 protein levels (post-transcriptional).
- Found a concomitant decrease in cdk6 mRNA and protein expression following MYCN suppression.
Conclusions:
- E2F and ID2 expression are significantly associated with MYCN regulation in neuroblastoma.
- CDK6 emerges as a potential novel transcriptional target of MYCN.
- These findings clarify MYCN's role in cell cycle control and identify potential therapeutic targets.
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