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Published on: September 14, 2016
[Establishment of nucleophosmin gene silenced HL-60 and its resistant cell line]
1Fujian Medical University Union Hospital, Fujian Institute of Hematology, Fujian Province, China.
Abstract:
This study was aimed to construct model cell line of NPM1-RNAi in HL-60 cells and its resistant line (HL-60/ADR) so as to provide a experimental basis for investigating the potential role of NPM1 gene in leukemia drug resistance. The shRNA targeting to NPM1 was ligated into linear pGCSIL-GFP vector, and transformed into E.coli DH5α. Positive clone was identified by PCR and DNA sequencing. pHelper 1.0, pHelper 2.0 and pGCSIL-GFP-NPM1-shRNA were cotransformed into 293T cells by lentivirus vector system. NPM1-RNAi-LV was transfected into HL-60 and HL-60/ADR cell lines. The efficiency of NPM-RNAi-LV was detected by using real-time quantitative RT-PCR and Western blot. The results showed that the recombinant eukaryotic expression vector pGCSIL-GFP-NPM1-shRNA was constructed. pGCSIL-GFP-NPM1-shRNA was packed into NPM1-RNAi-LV by lentivirus vector system, and transfected into HL-60 and HL-60/ADR cell lines. At mRNA level, the efficiency of NPM1 mRNA knockdown was more than 90% (p < 0.05). At protein level, obvious down-regulation of NPM protein was noted, indicating that NPM1 gene in HL-60 and HL-60/ADR cell lines was knocked down after transfected with NPM1-RNAi-LV. The resistance of HL-60/ADR cell line to adriamycin decreased to a certain degree after NPM1 gene silencing. It is concluded that the model cell lines of NPM1-RNAi in HL-60 and HL-60/ADR are successfully constructed, which can be used for investigating the potential role of NPM1 gene in drug resistance of leukemia.
Insights
Researchers created model cell lines to study how NPM1 gene impacts leukemia drug resistance. Silencing NPM1 reduced adriamycin resistance in HL-60/ADR cells, providing a basis for further leukemia research.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Leukemia drug resistance is a significant clinical challenge.
- The role of the NPM1 gene in leukemia drug resistance requires further investigation.
- Developing reliable cellular models is crucial for studying gene function in disease.
Purpose of the Study:
- To construct model cell lines of NPM1-RNAi in HL-60 and HL-60/ADR cells.
- To provide an experimental basis for investigating the role of the NPM1 gene in leukemia drug resistance.
- To assess the impact of NPM1 gene silencing on adriamycin resistance.
Main Methods:
- Construction of a recombinant eukaryotic expression vector (pGCSIL-GFP-NPM1-shRNA).
- Utilizing a lentivirus vector system for gene delivery into HL-60 and HL-60/ADR cell lines.
- Validation of NPM1 knockdown efficiency using real-time quantitative RT-PCR and Western blot.
Main Results:
- Successful construction and packaging of the NPM1-RNAi lentivirus vector.
- Achieved over 90% efficiency in NPM1 mRNA knockdown and significant NPM protein down-regulation.
- Demonstrated a partial decrease in adriamycin resistance in HL-60/ADR cells following NPM1 gene silencing.
Conclusions:
- Successfully established NPM1-RNAi model cell lines in HL-60 and HL-60/ADR.
- The constructed cell lines are suitable for investigating the role of NPM1 in leukemia drug resistance.
- NPM1 gene silencing shows a potential role in modulating leukemia drug resistance.
