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Evidence for miR-548c-5p regulation of FOXC2 transcription through a distal genomic target site in human podocytes

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Abstract

Podocytes are highly differentiated epithelial cells outlining the glomerular vessels. FOXC2 is a transcription factor essential for inducing podocyte differentiation, development and maturation, and is considered to be the earliest podocyte marker. miRNA prediction analysis revealed a full-length target site for the primate-specific miR-548c-5p at a genomic region > 8 kb upstream of FOXC2. We hypothesised that the transcription rates of FOXC2 during podocyte differentiation might be tuned by miR-548c-5p through this target site. Experiments were performed with cultured human podocytes, transfected with luciferase reporter constructs bearing this target site region within an enhancer element of the native plasmid. The results confirmed a seed region-driven targeting potential by the miRNA, with mimics downregulating and inhibitors enhancing luciferase activity. Introducing mutations into the miRNA target seed region abolished the expected response. In cultured podocytes, FOXC2 mRNA and protein levels responded to miR-548c-5p abundance in a coordinated manner before and after induction of differentiation, with high statistical significance. Ago-ChIP experiments revealed occupancy of the miRNA target site by miRNA/RISC in undifferentiated cells and its release when differentiation is initiated, allowing its interaction with the gene’s promoter region to amplify FOXC2 expression, as shown by chromosome conformation capture and qRT-PCR. Moreover, the expression pattern of FOXC2 during podocyte differentiation seems to be affected by miR-548c-5p, as removal of either endogenous or mimic miR-548c-5p results in increased FOXC2 protein levels and cells resembling those undergoing differentiation. Collectively, results indicate a well-orchestrated regulatory model of FOXC2 expression by a remote upstream target site for miR-548c-5p.

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Acknowledgements

The authors would like to thank Prof. Moin Saleem and Prof. Tobias Huber for providing the human and mouse podocyte cell lines, respectively. This project was supported by a grant co-funded by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation (Strategic Infrastructure Project NEW INFRASTRUCTURE/STRATEGIC/0308/24) to CD.

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Author notes

  1. Andrea Christofides and Gregory Papagregoriou contributed equally.

Authors and Affiliations

  1. Molecular Medicine Research Center and Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus

    Andrea Christofides, Gregory Papagregoriou & Constantinos Deltas

  2. The Wistar Institute, 3601 Spruce St, Philadelphia, PA, 19104, USA

    Harsh Dweep

  3. Developmental and Functional Genetics Group, Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

    Neoklis Makrides

  4. Medical Research Center, Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Germany

    Norbert Gretz

  5. Department of Life and Health Sciences, University of Nicosia, Nicosia, Cyprus

    Kyriacos Felekkis

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  1. Andrea Christofides
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Contributions

GP, AC, KF and CD designed the study; AC, GP and NM carried out experiments and analysed data; HD, NG and GP worked on predictions and bioinformatics, GP, AC, and CD drafted and revised the paper; all authors approved the final version of the manuscript.

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Correspondence to Gregory Papagregoriou or Constantinos Deltas.

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Supp. Table 1:

Primer sequences and product sizes (XLSX 12 kb)

Supp. Table 2:

Antibodies used and IF reagents (DOCX 14 kb)

Supp. Table 3:

Mean value comparison of predicted target sites on promoter regions between different miRNA species of family miR-548 (XLSX 23 kb)

Supp. Methods:

Supplementary methods (DOCX 16 kb)

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Christofides, A., Papagregoriou, G., Dweep, H. et al. Evidence for miR-548c-5p regulation of FOXC2 transcription through a distal genomic target site in human podocytes. Cell. Mol. Life Sci. 77, 2441–2459 (2020). https://doi.org/10.1007/s00018-019-03294-z

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