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Assessment of the Metabolic Profile of Primary Leukemia Cells
Published on: November 21, 2018
[Overexpression of miR-30a Promotes Apoptosis of Leukemia K562 Cells]
Min Xu1, Wen-Wan Gao1, Yu-Jie Luo1
1Department of Immunology, College of Basic Medical Science ,Chongqing Medical University, Chongqing 400016, China.
Objective:
To investigate the pro-apoptotic effect and mechanism of miR-30a overexpression on chronic myeloid leukemia K562 cells.
Methods:
The k562 cells were transfected with the recombinant plasmid pEGFP-pre-miR-30a, the real-time quantitative PCR was used to detect the level of miR-30a and BCR/ABL, and then the cell apoptosis was assessed by flow cytometry with AnnexinV-FITC/PI double staining. Western blot was used to detect the expression of BCR/ABL protein,apoptosis-related protein BCL-2 and BAX, PTEN, AKT and p-AKT.
Results:
Sequencing and digestion map indicated that the recombinant plasmid was constructed successfully. Compared with 2 control groups, the miR-30a expression in k562 cells transfected with recombinant plasmid pEGFP-pre-miR-30a was obviously up-regulated. The expression of BCR/ABL mRNA and BCR/ABL protein was both significantly down-regulated. Apoptotic rate was significantly enhanced (both P<0.05),and the expression of anti-apoptotic protein BCL-2 was down-regulated while the expression of pro-apoptotic protein BAX was up-regulated. The level of PTEN was significantly up-regulated in omparison with control groups,no variation was found in total AKT, but the expression of p-AKT was down-regulated.
Conclusion:
The overexpression of miR-30a is abled to down-regulate the level of BCR/ABL mRNA and BCR/ABL protein, and increase apoptotic rate, its mechanism may be related with inhibition of the activity of BCR/ABL-PTEN/AKT signaling pathway.
Insights
Overexpressing miR-30a in chronic myeloid leukemia cells triggers apoptosis by down-regulating BCR/ABL and inhibiting the BCR/ABL-PTEN/AKT pathway.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- Chronic myeloid leukemia (CML) is characterized by the BCR/ABL fusion gene.
- Understanding novel therapeutic targets for CML is crucial.
- MicroRNAs play significant roles in cancer development and progression.
Purpose of the Study:
- To investigate the pro-apoptotic effects of miR-30a overexpression in K562 CML cells.
- To elucidate the underlying molecular mechanisms, including the BCR/ABL-PTEN/AKT signaling pathway.
Main Methods:
- K562 cells were transfected with a miR-30a precursor plasmid.
- Real-time quantitative PCR (RT-qPCR) assessed miR-30a and BCR/ABL levels.
- Flow cytometry analyzed apoptosis.
- Western blotting detected protein expression of BCR/ABL, BCL-2, BAX, PTEN, AKT, and phosphorylated AKT (p-AKT).
Main Results:
- miR-30a expression was significantly upregulated post-transfection.
- BCR/ABL mRNA and protein levels were significantly downregulated.
- Apoptotic rates increased significantly, with decreased BCL-2 and increased BAX.
- PTEN expression increased, while p-AKT levels decreased, indicating inhibited AKT signaling.
Conclusions:
- miR-30a overexpression induces apoptosis in K562 cells.
- This effect is mediated by downregulating BCR/ABL and inhibiting the BCR/ABL-PTEN/AKT signaling pathway.
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