Functional interaction of microRNAs with chemokine ligands and their expression in the microenvironment of colorectal tumors

Einleitung/Background:

MicroRNAs (miRNAs) are naturally occurring short noncoding RNAs that regulate gene expression at the post-transcriptional level and play a role in cancer pathogenesis. Recently, involvement of chemokine CCL20 and its receptor CCR6 in colorectal cancer (CRC) progression was shown.

Ziele/Aims:

Here, we analyzed the functional interaction of two miRNA candidates with CCL20 and their impact on CCL20 expression in CRC cells.

Methodik/Methods:

Various prediction software tools were applied to identify miRNAs which potentially interact with CCL20. Functional implications of these miRNAs with the 3'UTR of CCL20 were analyzed using the Dual Luciferase Reporter assay system. Three CRC cell lines were transfected with miR-21 miRNA mimics and gene and protein expression of CCL20 was monitored using qRT PCR and ELISA, respectively. For the presentation of miRNA and chemokine cellular localization in situ hybridization and immunohistochemistry were applied.

Ergebnis/Results:

MiR-21 and miR-145 were identified to potentially interact with CCL20 by computer software. Subsequently, only addition of miR-21 led to a significant down-regulation of luciferase activity, which was significantly reversed in a reporter test system containing the mutated seed sequence in the 3'UTR of CCL20. Following transfection of CRC cell lines with miR-21 mimics and subsequent monitoring of CCL20 expression showed significant down-regulation of CCL20 mRNA and protein expression. MiR-21 expression was located on the cellular level predominantly in stromal cells like tumor-associated fibroblasts and to a minor degree in immune cells like macrophages and lymphocytes. Likewise, CCL20 expression was primarily detected in tumor-infiltrating immune cells.

Schlussfolgerung/Conclusions:

As we demonstrated a functional interaction of miR-21 with its target CCL20 in various CRC cell lines, we speculated that regulation of CCL20 expression by miR-21 might be a regulatory mechanism involved in progression of CRC. However, investigating their cellular localization revealed that both molecules are expressed predominantly in the microenvironment of CRC tumors which may be explained by the tumor-conducted attraction of immune cells to the tumor microenvironment.