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Published on: February 27, 2015
Identification of miR-21 targets in breast cancer cells using a quantitative proteomic approach
Yi Yang1, Raghothama Chaerkady, Michael A Beer
1McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
Abstract:
MicroRNA (miRNA) play essential roles in biological processes ranging from cellular proliferation to apoptosis. Recently, miRNA have also been implicated in a number of diseases including cancers. However, the targets of most miRNA remain unknown. The majority of reports describing identification of miRNA targets are based on computational approaches or detection of altered mRNA levels despite the fact that most miRNA are thought to regulate their targets primarily at the level of translational inhibition in animals. miR-21 is a miRNA with oncogenic activity that is involved in various cancer-related processes such as invasion and migration. Given the importance of miR-21 in tumorigenesis, we employed a quantitative proteomic strategy to systematically identify potential targets of miR-21. By knocking down the expression of endogenous miR-21 in MCF-7 breast cancer cells, we observed an increase in the abundance of 58 proteins, implying that they could be potential targets of miR-21. Validation of 12 of these candidate targets in luciferase assays showed that 6 of them were likely direct targets of miR-21. Importantly, the mRNA of the majority of the candidate targets tested did not show a concomitant increase in abundance. Overall, our results demonstrate that miR-21 affects the expression of many of its targets through translational inhibition and highlights the utility of proteomic approaches for identifying miRNA targets.
Insights
This study identifies microRNA-21 (miRNA-21) targets in breast cancer cells using quantitative proteomics. Researchers found that miRNA-21 primarily inhibits protein translation, not mRNA levels, for many of its targets.
Area of Science:
- Molecular Biology
- Cancer Research
- Proteomics
Background:
- MicroRNAs (miRNAs) are crucial regulators of cellular processes and are implicated in diseases like cancer.
- Identifying miRNA targets is challenging, as most regulation occurs via translational inhibition, not mRNA degradation.
- miR-21 is an oncogenic miRNA involved in cancer progression, making its targets critical for understanding tumorigenesis.
Purpose of the Study:
- To systematically identify direct protein targets of the oncogenic microRNA-21 (miR-21).
- To investigate the mechanism of miR-21-mediated gene regulation, focusing on translational inhibition versus mRNA degradation.
- To demonstrate the utility of quantitative proteomic strategies in discovering miRNA targets.
Main Methods:
- Utilized a quantitative proteomic approach in MCF-7 breast cancer cells with endogenous miR-21 knockdown.
- Identified proteins with increased abundance upon miR-21 depletion as potential targets.
- Validated candidate targets using luciferase assays and assessed corresponding mRNA level changes.
Main Results:
- Knockdown of miR-21 led to increased abundance of 58 proteins, suggesting they are miR-21 targets.
- Luciferase assays confirmed 6 out of 12 tested proteins as direct targets of miR-21.
- The mRNA levels of most validated targets did not significantly increase, supporting translational inhibition as the primary mechanism.
Conclusions:
- miR-21 regulates the expression of numerous targets predominantly through translational inhibition.
- Proteomic strategies are effective for identifying miRNA targets, especially when translational regulation is involved.
- This study provides a validated list of miR-21 targets relevant to breast cancer progression.
