Related Experiment Video
Updated: Oct 3, 2026
Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs
Published on: May 15, 2019
[Effects of PML and PML-RAR alpha antisense oligonucleotides on promyelocytic leukemia cell line NB4]
1Shanghai Institute of Hematology, Renji Hospital, Laboratory of Leukemia Research, Shanghai Second Medical University, Shanghai 200001, China.
Objective:
To investigate the different effects of anti-PML (promyelocytic leukemia) and anti-PML/RAR alpha (promyelocytic leukemia/retionic acid receptor alpha) antisense oligonucleotides on cell growth, expression of PML-RAR alpha mRNA and PML-RAR alpha/PML protein location of NB4 cell line.
Methods:
RT-PCR was used for PML-RAR alpha mRNA expression, trypan blue exclusion for cell count, methylcellulose assay for leukemic colony forming unit, immuno-fluorescence for PML-RAR alpha/PML protein localization.
Results:
Both anti-PML start codon region antisense (STAS) and anti-PML-RAR alpha fusion region antisense (FUAS) could inhibit cell growth and formation AML-CFU. Cells became partially differentiated on day 5, being more marked in FUAS-treated cells than in STAS-treated ones. Down regulated PML-RAR alpha mRNA expression occurred at 24 h was in STAS and FUAS-treated cells and maintained for up to 72 h. Immuno-fluorescence analysis with anti-PML monoclonal antibody showed a remarkable decrease to almost complete disappearance of microgranules. The residual granules became enlarged to become discrete dots (< 10 per cell), similar to normal POD structure in some STAS-treated cells at 24 h. AT 72 h, nearly all the granules disappeared. Similar changes were observed in FUAS-treated cells.
Conclusion:
Both PML and PML-RAR alpha antisense oligonucleotides can specifically block the expression of PML-RAR alpha at mRNA and protein levels. PML protein is implicated in the regulation of cell differentiation.
Insights
Antisense oligonucleotides targeting PML and PML-RAR alpha inhibit NB4 cell growth and differentiation. Both treatments effectively reduced PML-RAR alpha mRNA and protein levels, impacting leukemic cell formation.
Area of Science:
- Molecular Biology
- Cell Biology
- Hematology
Background:
- Acute Promyelocytic Leukemia (APL) is characterized by the PML-RAR alpha fusion gene.
- Antisense technology offers a targeted approach to gene expression modulation.
Purpose of the Study:
- To compare the effects of anti-PML and anti-PML/RAR alpha antisense oligonucleotides on NB4 cells.
- To assess the impact on cell growth, differentiation, and gene/protein expression.
Main Methods:
- Reverse Transcription Polymerase Chain Reaction (RT-PCR) for mRNA analysis.
- Trypan blue exclusion assay for cell viability.
- Methylcellulose assay for colony formation.
- Immunofluorescence for protein localization.
Main Results:
- Both anti-PML (STAS) and anti-PML/RAR alpha (FUAS) inhibited NB4 cell growth and leukemic colony formation.
- Partial cell differentiation was observed, more pronounced with FUAS.
- PML-RAR alpha mRNA and protein levels were significantly downregulated by both treatments.
- PML protein localization showed changes, with granule disappearance and formation of discrete dots.
Conclusions:
- PML and PML-RAR alpha antisense oligonucleotides effectively inhibit gene expression at mRNA and protein levels.
- PML protein plays a role in regulating cell differentiation in APL.
