1 Department of Clinical Medicine - Molekylær Medicinsk afdeling (MOMA), Department of Clinical Medicine, Health, Aarhus University2 Department of Genetics and Pathology Pomeranian Medical University, Szczecin3 Ludwig Colon Cancer Initiative Laboratory, Ludwig Institute for Cancer Research4 Department of Clinical Medicine - Molekylær Medicinsk afdeling (MOMA), Department of Clinical Medicine, Health, Aarhus University
Colorectal cancer (CRC) is one of the leading causes of cancer deaths. Twenty-five percent of the patients radically treated for a stage II CRC (no lymph node or distant metastasis) later develop recurrence and dies from the disease. MicroRNAs (miRNAs) are aberrantly expressed or mutated in human cancers, and function either as tumour suppressors or oncogenes. Additionally, they also appear to have both diagnostic and prognostic significance. The aim of the present study was to identify miRNAs associated with recurrence of stage II CRC, followed up by an investigation of how these potential biomarkers functionally influence tumour cell behaviour. TaqMan® Human MicroRNA Array Set v2.0 was use to profile the expression of more than 600 miRNAs in 46 stage II CRC tumours (23 without recurrence and 23 with recurrence). Four miRNAs; were identified as being associated with recurrence of the disease. Kaplan Meier analysis showed significant correlations between low expression of the four miRNAs and poor prognosis. Functional characterization of their impact on cell viability using MTT analysis demonstrated that they all inhibit the viability of HCT116 cells. One miRNAs also inhibited the viability of DLD1, LS174T and SW480 cells and was selected for further analysis. In order to get an understanding of the mechanism by which the miRNA affect cellular growth it was decided to elucidate its target network. Candidate mRNAs targets were identified using an integrative approach combining in silico target prediction and transcript profiling. Initially, miRNA over-expression in HCT116 cells was followed by transcriptional profiling of transfected cells using GeneChip Human Exon 1.0 ST Arrays. Three in silico predicted miRNA targets showing differential mRNA expression upon miRNA up-regulation were selected for detailed analysis. Luciferase miRNA target reporter assays confirmed all three mRNAs to be direct targets. Secondly, siRNA mediated knock-down of the potential targets resulted in growth suppression of HCT116 cells, mimicking the effect of the miRNA. In conclusion, miRNAs are associated with recurrence of stage II CRC and has the capacity to inhibit cellular growth in vitro through binding to specific targets.