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Updated: Jun 25, 2026
An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
Two new miR-16 targets: caprin-1 and HMGA1, proteins implicated in cell proliferation
Tagrid Kaddar1, Jean-Pierre Rouault, Wei Wen Chien
1LBMC, CNRS UMR5239, IFR128, Faculté de Médecine Lyon Sud, Université Lyon 1, Pierre Bénite, France.
Background Information:
miRNAs (microRNAs) are a class of non-coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3' UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR-16 (miRNA-16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR-16.
Results:
In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR-16, caprin-1 (cytoplasmic activation/proliferation-associated protein-1) and HMGA1 (high-mobility group A1), and we also studied cyclin E which had been previously recognized as an miR-16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR-16 interacts with the 3' UTR of the three target mRNAs. We showed that miR-16, in MCF-7 and HeLa cell lines, down-regulates the expression of caprin-1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels.
Conclusions:
Taken together, our data demonstrated that miR-16 can negatively regulate two new targets, HMGA1 and caprin-1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.
Insights
MicroRNA-16 (miR-16) down-regulates cell proliferation targets, including high-mobility group A1 (HMGA1) and cytoplasmic activation/proliferation-associated protein-1 (caprin-1). This study validates HMGA1 and caprin-1 as novel miR-16 targets, impacting cell growth pathways.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by targeting messenger RNAs (mRNAs).
- miR-16, a specific miRNA, is implicated in cell proliferation, apoptosis, and leukaemogenesis.
- Identifying novel miRNA targets is crucial for understanding complex gene regulatory networks.
Purpose of the Study:
- To identify and validate novel targets of miR-16.
- To investigate the role of miR-16 in regulating cell proliferation-associated genes.
- To elucidate the mechanisms by which miR-16 affects target gene expression.
Main Methods:
- Bioinformatics analysis to predict potential miR-16 targets.
- Luciferase activity assays to confirm direct interaction of miR-16 with target 3' UTRs.
- Western blot and quantitative PCR to assess protein and mRNA level changes.
Main Results:
- Two novel miR-16 targets, caprin-1 and HMGA1, were identified and validated.
- miR-16 was shown to directly bind to the 3' UTR of caprin-1, HMGA1, and cyclin E mRNAs.
- Down-regulation of caprin-1, HMGA1 (isoforms a and b), and cyclin E at protein and mRNA levels was observed in response to miR-16.
Conclusions:
- miR-16 negatively regulates HMGA1 and caprin-1, both involved in cell proliferation.
- The observed inhibition of cyclin E expression is partly attributed to decreased mRNA stability.
- These findings expand the understanding of miR-16's role in cellular processes and disease.
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