IDENTIFICATION OF INTRONIC MICRORNAS ALTERED IN BREAST CANCER THROUGH MICROARRAY HOST GENE EXPRESSION ANALYSIS

MicroRNA (miRNAs) are endogenous non-coding RNAs of ∼ 22 nucleotides in length that function as post-transcriptional regulators of gene and pro- tein expression through degradation or translation inhibition of the target messenger RNAs. MiRNAs show altered expression profiles in several hu- man pathologies, including cancer. They can act as tumour suppressors or as oncogenes, depending on the characteristics of their target genes. More than half of the mammalian miRNAs, including several of the miR- NAs implicated in breast cancer, are localized within the introns of protein- coding genes, often organized in clusters, and usually transcribed together with their host gene. It is therefore possible, at least in principle, to iden- tify novel intronic cancer-regulated miRNAs by examining the expression profile of their host genes by means of microarrays. For this purpose, we analyzed the regulation of 253 miRNA host genes in five large breast cancer microarray data sets comprising more than 950 samples, examining their association with different clinical and pathological parameters such as tu- mour grade, estrogen and progesterone receptor status, p53 status, survival, and occurrence of relapse or metastasis. We found that MCM7 and SMC4 were the most frequently and significantly overexpressed genes in high grade tumours. These genes contain two well known cancer-associated miRNA clusters: miR25-93-106b and miR-15b/16- 2 respectively. In addition we identified six other miRNA host genes that were significantly downregulated in high grade tumours in all the data sets. Much less evidence is available in the literature about the involvement in cancer of the miRNAs contained in these genes (i.e., miR-218-1, miR-342, miR-483, miR-548f-2, miR-1245 and miR-1266 ). We measured the expression of the selected miRNAs by Real Time PCR on an independent cohort of 36 formalin-fixed paraffin-embedded (FFPE) samples, and we observed reduced expressions level of such miRNAs in high grade tumours. In particular, we found miR-342-3p, miR-342-5p, miR-483- 3p, and miR-483-5p to be the most significantly downregulated miRNAs. These miRNAs were also found to correlate with bad prognosis in grade 2 tumours. Finally we provided initial evidence that increased expression of miR-342-5p, but not miR-342-3p, induces apoptosis in the highly metastatic MDA-MB-231 breast cancer cell line.