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Proliferation of metanephric mesenchymal cells is inhibited by miR-743a-mediated WT1 suppression in vitro
Mingming Xue1, Yuru Zhou2, Xiaoyu Liu1
1Division of Molecular Nephrology and The Creative Training Center for Undergraduates, M.O.E. Key Laboratory of Laboratory Medical Diagnostics, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, P.R. China.
Abstract:
To seek out the potential microRNAs (miRNAs) that target Wilms' tumor suppressor 1 (WT1), a transcription factor required for progenitor proliferation as well as normal development of the kidney, and to clarify the effects of the miRNAs on WT1, the 3'-untranslated region (3'‑UTR) of WT1 was initially analyzed and miR‑743a, a seldom‑reported miRNA, was identified. In the present paper, luciferase reporter assays were performed to confirm that miR‑743a is able to directly target the 3'‑UTR of WT1. Subsequently, reverse transcription‑quantitative polymerase chain reaction, combined with western blotting analyses, were performed, and the results revealed a significant inhibition of WT1 at the mRNA and the protein levels. Furthermore, a 5‑ethynyl‑2'‑deoxyuridine (EdU) cell proliferation assay, coupled with a WT1 rescue strategy, demonstrated that miR‑743a inhibited the proliferation of metanephric mesenchymal (MM) cells, in part by targeting WT1. In conclusion, by targeting WT1, miR‑743a suppresses the proliferation of MM cells in vitro, and probably possesses vital functions in kidney development and kidney‑associated diseases.
Insights
MicroRNA-743a (miR-743a) targets Wilms' tumor suppressor 1 (WT1), inhibiting kidney progenitor cell proliferation. This finding is crucial for understanding kidney development and associated diseases.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Wilms' tumor suppressor 1 (WT1) is essential for kidney development and progenitor cell proliferation.
- MicroRNAs (miRNAs) play regulatory roles in gene expression, impacting various biological processes.
Purpose of the Study:
- To identify miRNAs targeting WT1.
- To elucidate the functional impact of identified miRNAs on WT1 expression and kidney cell proliferation.
Main Methods:
- Analysis of WT1 3'-untranslated region (3'-UTR).
- Luciferase reporter assays to confirm miRNA targeting.
- Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting to assess mRNA and protein levels.
- 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay with WT1 rescue experiments.
Main Results:
- miR-743a was identified as a direct targeting miRNA for WT1.
- miR-743a significantly inhibited WT1 mRNA and protein expression.
- miR-743a suppressed metanephric mesenchymal (MM) cell proliferation, partly through WT1 targeting.
Conclusions:
- miR-743a suppresses MM cell proliferation in vitro by targeting WT1.
- miR-743a likely plays a significant role in kidney development and kidney-related diseases.
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