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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
Micro-RNA-21 regulates the sensitivity to cisplatin in human neuroblastoma cells
Yun Chen1, Ya-Hui Tsai, Yu Fang
1Department of Surgery, Far Eastern Memorial Hospital, Pan-Chiao, New Taipei, Taiwan.
Background/Purpose:
Drug resistance often causes treatment failure in neuroblastomas. Increasing evidence has implicated that the micro-RNAs (miRNAs) are involved in the development of drug resistance. In this report, we aimed to investigate the role of miRNA in cisplatin resistance of neuroblastoma cells.
Materials And Methods:
The cell viability of the neuroblastoma cells after cisplatin treatment was analyzed. The expression of the miRNAs and phosphatase and tensin homolog (PTEN) messenger RNA in the neuroblastoma cells was studied by real-time polymerase chain reaction. Overexpression of miRNA or suppression of miRNA expression by antagomir was used to investigate the effects of miRNA on the cisplatin-induced cell death or proliferation.
Results:
The expression of miR-21 was increased in the cisplatin-resistant (CisR) neuroblastoma cells as compared with the parental cells, and the antagomir against miR-21 converted the resistant cells into sensitive ones. Ectopic expression of pre-miR-21 in parental cells resulted in decreased sensitivity to cisplatin treatment. In addition, overexpression of pre-miR-21 markedly increa sed the proliferation rate of neuroblastoma cells. The level of PTEN messenger RNA and protein in the CisR cells was lower than that in the parental cells. Transfection of pre-miR-21 into the parental cells reduced the PTEN expression, and transfection of anti-miR-21 into the CisR cells increased the PTEN expression.
Conclusion:
Micro-RNA-21 regulated the drug resistance and proliferation in neuroblastoma cells.
Insights
Micro-RNA-21 (miRNA-21) promotes cisplatin resistance and proliferation in neuroblastoma cells by downregulating PTEN. Inhibiting miRNA-21 can restore sensitivity to chemotherapy.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Drug resistance is a major cause of treatment failure in neuroblastoma.
- Micro-RNAs (miRNAs) are increasingly recognized for their role in drug resistance development.
Purpose of the Study:
- To investigate the specific role of miRNA in conferring cisplatin resistance in neuroblastoma cells.
- To elucidate the molecular mechanisms underlying miRNA-mediated drug resistance.
Main Methods:
- Real-time PCR was used to analyze miRNA and PTEN mRNA expression.
- Cell viability assays were performed post-cisplatin treatment.
- miRNA overexpression and antagomir inhibition were employed to assess functional effects.
Main Results:
- miR-21 expression was elevated in cisplatin-resistant (CisR) neuroblastoma cells.
- miR-21 inhibition resensitized CisR cells to cisplatin, while its overexpression decreased sensitivity in parental cells.
- PTEN levels were inversely correlated with miR-21 expression, suggesting PTEN is a target.
Conclusions:
- Micro-RNA-21 plays a critical role in regulating cisplatin resistance and cell proliferation in neuroblastoma.
- Targeting miR-21 presents a potential therapeutic strategy to overcome drug resistance in neuroblastoma.
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