Chemokine receptor CCR6 expression is regulated by miR-518a-5 p in colorectal cancer cells

Einleitung:

Recently, involvement of the chemokine/receptor system CCL20/CCR6 in colorectal cancer (CRC) progression was shown.

Ziele:

Here, we analyzed the functional interaction of miRNA-518 (miR-518a-5 p) with CCR6 and its impact on CCR6 expression in CRC cells.

Methodik:

MiR-518a-5 p was identified by computer software to potentially interact with CCR6. Hence, functional implications of miR-518a-5 p with the 3'UTR of CCR6 were analyzed using the Dual Luciferase Reporter assay system. Confirmation of the predicted target site for miR-518a-5 p was achieved by site-directed mutagenesis of the seed sequence in the 3'UTR of CCR6 and subsequent application of the mutated seed sequence in a luciferase assay with miR-518a-5 p mimics. Accordingly, two CRC cell lines (Caco-2 and HT-29) were transfected with miR-518a-5 p miRNA mimics and gene and protein expression of CCR6 was monitored using qRT PCR and immunocytochemistry, respectively.

Ergebnisse:

Addition of miR-518a-5 p led to significant down-regulation of luciferase activity (P < 0,05), which was significantly reversed in a reporter test system containing the mutated seed sequences in the 3'UTR of CCR6. Following transfection of CRC cell lines with miR-518a-5 p mimics and subsequent monitoring of CCR6 expression showed significant down-regulation of CCR6 mRNA and CCR6 protein expression in both CRC cell lines under investigation (P < 0,05).

Schlussfolgerung:

We have shown that miR-518a-5 p functionally interacts with CCR6 and that transfection of CRC cells with miR-518a-5 p leads to significant CCR6 down-regulation. Consequently, CCR6 expression is regulated by miR-518a-5 p in CRC cells indicating that regulation of CCR6 expression by miR-518a-5 p might be a regulatory mechanism involved in CRC pathogenesis.