miR-224 is marker of poor prognosis in breast cancer.


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Sharon McGee (1), Laoighse Mulrane (1), Donal Brennan (1), Susan McDonnell (1), Karin Jirstrom (2), Darran O'Connor (1), William Gallagher (1)
University College Dublin, Dublin, Ireland (1), Lund University, Lund, Sweden (2)

Background

Breast cancer is the most common cancer in women and, after lung cancer, the second leading cause of cancer-related deaths. MicroRNAs (miRNAs) are highly conserved non-coding RNA molecules that regulate gene expression by binding the 3’ untranslated region of mRNA causing degradation or inhibition of translation. Thus by regulating protein expression at the mRNA level, miRNAs have the ability to induce changes in multiple pathways and processes. Here, we examine a tractable cell line model of breast cancer progression to identify miRNAs involved.

Method

Total RNA from Hs578T breast cancer cells and an isogenic super-invasive variant Hs578T(I8)1 was isolated and miRNAs were reverse transcribed using miRNA-specific primers (Applied Biosystems). miRNA profiling was subsequently performed using an Early Access Human miRNA TaqMan Assay (Applied Biosystems). Differentially expressed miRNAs were identified by the DDCT method, with normalisation achieved using 4 endogenous controls identified by geNORM. Finally, the miRNA with the greatest expression in the super-invasive cell line was evaluated in a cohort of breast cancer patients (n=98), via locked nucleic acid in situ hybridisation (ISH) on tissue microarrays (TMAs).

Results

Eight miRNAs were found to be differentially expressed between Hs578T breast cells and their super-invasive derivative, with a minimum fold change of 2 and a minimal cycle threshold of 32. Of these, miR-224 was most significantly up-regulated in the invasive derivative. Subsequent ISH analysis by TMA and automated image analysis of its expression in breast tumours found that high miR-224 expression correlated with poor overall survival in the entire cohort (p=0.022). Furthermore, high miR-224 expression was associated with poor overall survival in ER-positive (p=0.015) and Her2-negative (p=0.008) patients (HR 4.107, p = 0.015, 95% CI 1.322-12.758).

Conclusion

We thus believe that miR-224 represents a novel marker of poor prognosis in breast cancer that may regulate breast cancer invasiveness and disease progression.

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