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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
MiR-1180-5p regulates apoptosis of Wilms' tumor by targeting p73
1Neonatal Intensive Care Unit, Zhoukou Central Hospital, Zhoukou.
Introduction:
Wilms' tumor (WT), the most common childhood tumor, occurs in sporadic or familial forms. Recent findings reported that abnormal expression in microRNA (miRNA) suggests an important role of miRNAs during WT progress. MiRNAs are endogenous short-chain noncoding RNAs, which have been reported as key biomarkers for detecting tumor onset and progression. However, the functional role of miR-1180 in WT has remained unknown.
Materials And Methods:
MTT and clonogenic survival assays were used to detect WT cell proliferation. Flow cytometry Annexin V-FITC was used to measure apoptosis. In addition, proteins expressions in the cells were determined by Western blotting.
Results:
In the present study, we demonstrated that miR-1180 is upregulated in WT when compared with adjacent tissues by quantitative reverse-transcription polymerase chain reaction. In addition, the inhibition of miR-1180 induced apoptosis in SK-NEP-1 cell line in vitro. Moreover, luciferase reporter assay showed that p73 protein was the target of miR-1180, which was confirmed by the results of Western blotting. Finally, in vivo data indicated that the tumor growth in mice was significantly inhibited by miR-1180 inhibitor.
Conclusion:
Our results indicate that miR-1180 might serve as a therapeutic target for future WT therapy.
Insights
MicroRNA-1180 (miR-1180) is upregulated in Wilms' tumor (WT). Inhibiting miR-1180 induces apoptosis and suppresses tumor growth, suggesting miR-1180 as a potential therapeutic target for WT.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Wilms' tumor (WT) is the most common childhood cancer.
- MicroRNAs (miRNAs) play a role in WT development and progression.
- The specific function of miR-1180 in WT remains unclear.
Purpose of the Study:
- To investigate the role of miR-1180 in Wilms' tumor.
- To determine if miR-1180 can be a therapeutic target for WT.
Main Methods:
- Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure miR-1180 expression.
- In vitro assays (MTT, clonogenic survival, flow cytometry) to assess cell proliferation and apoptosis.
- Western blotting and luciferase reporter assays to identify miR-1180 targets.
- In vivo studies in mice to evaluate the effect of miR-1180 inhibition on tumor growth.
Main Results:
- miR-1180 expression is significantly upregulated in WT tissues compared to adjacent tissues.
- Inhibition of miR-1180 induces apoptosis in WT cells (SK-NEP-1 cell line) in vitro.
- p73 protein was identified as a direct target of miR-1180.
- Inhibition of miR-1180 significantly suppressed tumor growth in a mouse model.
Conclusions:
- miR-1180 is upregulated in Wilms' tumor and contributes to tumor progression.
- Targeting miR-1180 may represent a novel therapeutic strategy for Wilms' tumor.
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