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Transcription factor FOXD1 and miRNA-204-5p play a major role in B4GALNT2 downregulation in colon cancer
Martina Duca1, Nadia Malagolini1, Michela Pucci1
1Department of Medical and Surgical Sciences (DIMEC), General Pathology Building, University of Bologna, Bologna, Italy.
Abstract:
The β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) which synthesizes the histo-blood group antigen Sda is highly expressed by normal colon, but it is dramatically down-regulated in colorectal cancer (CRC). High B4GALNT2 expression in CRC tissues is a marker of longer survival. The molecular bases of B4GALNT2 inhibition in CRC are largely obscure. A key role may be played by transcription factors and miRNA. Through an in silico analysis of The Cancer Genome Atlas and of the Cancer Cell Line Encyclopedia, we identified the transcription factors FOXD1, FOXF2 and PGR as well as mir-204-5p as potential inhibitory agents. Their transient transfection in the cell line GP2d, whose B4GALNT2 is closer to that of a normal mucosa, confirmed their inhibitory activity with a crucial role for FOXD1. The latter inhibited B4GALNT2 also in the middle B4GALNT2 expresser cell line Caco2. Deletion experiments of the putative FOXD1 binding sites in the ~ 2800 bp sequence upstream of the B4GALNT2 transcriptional start site cloned in frame with the luciferase reporter gene, confirmed the regulatory role of FOXD1. Finally, FOXD1 knock down in the non-B4GALNT2 expresser cell line SW948 stimulated B4GALNT2. Thus, FOXD1 and miR-204-5p emerged as crucial new player of B4GALNT2 down-regulation in CRC.
Insights
Transcription factors FOXD1 and miR-204-5p significantly inhibit β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) expression. This finding is crucial for understanding colorectal cancer progression and B4GALNT2 down-regulation.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) synthesizes the Sda antigen and is downregulated in colorectal cancer (CRC).
- High B4GALNT2 expression in CRC correlates with better patient survival, but the mechanisms of its inhibition are unclear.
- Transcription factors and microRNAs are potential regulators of B4GALNT2 expression.
Purpose of the Study:
- To identify molecular factors responsible for the down-regulation of B4GALNT2 in colorectal cancer.
- To investigate the roles of transcription factors and microRNAs in regulating B4GALNT2 expression.
Main Methods:
- In silico analysis of The Cancer Genome Atlas and Cancer Cell Line Encyclopedia to identify potential regulatory factors.
- Transient transfection assays in cell lines (GP2d, Caco2, SW948) to validate the inhibitory effects of identified factors.
- Reporter gene assays using luciferase to confirm FOXD1 binding site functionality.
- FOXD1 knockdown experiments to assess its impact on B4GALNT2 expression.
Main Results:
- FOXD1, FOXF2, PGR, and miR-204-5p were identified as potential inhibitors of B4GALNT2.
- Transient transfection confirmed the inhibitory activity of these factors, with FOXD1 showing a crucial role.
- FOXD1 demonstrated inhibitory effects on B4GALNT2 in multiple cell lines.
- Deletion of putative FOXD1 binding sites abolished FOXD1's regulatory effect.
- FOXD1 knockdown led to increased B4GALNT2 expression in a non-expressing cell line.
Conclusions:
- FOXD1 and miR-204-5p are key regulators involved in the down-regulation of B4GALNT2 in colorectal cancer.
- These findings provide new insights into the molecular mechanisms underlying B4GALNT2 regulation in CRC.
- FOXD1's role as a direct inhibitor of B4GALNT2 has been confirmed through functional studies.
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