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Identification of Cellular Targets of MicroRNA-181a in HepG2 Cells: A New Approach for Functional Analysis of
Jane Yi Lin Tan1, Nagy A Habib2, York Wieo Chuah3
1School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore, Singapore.
Abstract:
MicroRNAs (miRNAs) are known to play a part in regulating important cellular processes. They generally perform their regulatory function through their binding with mRNAs, ultimately leading to a repression of target protein expression levels. However, their roles in cellular processes are poorly understood due to the limited understanding of their specific cellular targets. Aberrant levels of miRNAs have been found in hepatocellular carcinoma (HCC) including miR-181a. Using bioinformatics analysis, cyclin-dependent kinase inhibitor 1B (CDKN1β) and transcriptional factor E2F7 were identified as potential targets of miR-181a. Validation analysis using surface plasmon resonance (SPR) showed a positive binding between miR-181a and the 3'UTRs of these two potential mRNA targets. In vivo luciferase assay further confirmed the positive miR-181a:mRNA bindings, where a significant decrease in luciferase activity was detected when HepG2 cells were co-transfected with the 3'UTR-containing reporter plasmids and miR-181a. The potential impact of miR-181a binding to its specific targets on the general cellular behavior was further investigated. Results showed that miR-181a significantly activated the MAPK/JNK pathway which regulates cell proliferation, supporting our recently reported findings. Inhibition of miR-181a, on the other hand, abolished the observed activation. Our findings open up a new approach in designing targeted functional analysis of miRNAs in cellular processes, through the identification of their cellular targets.
Insights
MicroRNAs regulate cellular processes by binding mRNA. This study identifies miR-181a targets, cyclin-dependent kinase inhibitor 1B and E2F7, revealing its role in hepatocellular carcinoma and MAPK/JNK pathway activation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) are key regulators of cellular processes, primarily through mRNA binding and subsequent protein expression repression.
- Understanding specific miRNA targets is crucial for elucidating their roles in cellular functions, yet remains a significant knowledge gap.
- Aberrant miRNA expression, including miR-181a, is implicated in diseases like hepatocellular carcinoma (HCC).
Purpose of the Study:
- To identify specific mRNA targets of miR-181a using bioinformatics analysis.
- To experimentally validate the binding interactions between miR-181a and its predicted targets.
- To investigate the functional consequences of miR-181a targeting on cellular pathways, specifically the MAPK/JNK pathway.
Main Methods:
- Bioinformatics analysis to predict potential mRNA targets of miR-181a.
- Surface Plasmon Resonance (SPR) to validate direct binding between miR-181a and the 3'UTRs of candidate mRNA targets.
- In vivo luciferase assay in HepG2 cells to confirm miR-181a:mRNA interactions.
- Investigation of the MAPK/JNK signaling pathway activation in response to miR-181a expression and inhibition.
Main Results:
- Cyclin-dependent kinase inhibitor 1B (CDKN1β) and transcriptional factor E2F7 were identified as potential targets of miR-181a.
- SPR and luciferase assays confirmed direct binding of miR-181a to the 3'UTRs of CDKN1β and E2F7 mRNA.
- miR-181a significantly activated the MAPK/JNK pathway, which is involved in cell proliferation; inhibition of miR-181a abolished this activation.
Conclusions:
- This study successfully identified CDKN1β and E2F7 as direct targets of miR-181a.
- The findings demonstrate miR-181a's role in activating the MAPK/JNK pathway, impacting cell proliferation.
- The identification of specific miRNA targets provides a foundation for targeted functional analyses and potential therapeutic strategies in diseases like HCC.
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