Frequency of Hsa-miR9 aberrant methylation in metastatic breast cancers.

MicroRNAs (miRNAs) are a recently discovered class of very small non-coding RNAs involved in the regulation of gene expression by interfereing with mRNA translation. It has been shown that human miR-9 expression levels are reduced in breast cancer samples due to the aberrant methylation of its promoter region. Methods: We analyzed74breast cancer cases treated by surgery at the IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy. Pathological assessment included evaluation of histological type, grade and stage. Estrogen receptor (ER), progesterone receptor (PgR), Ki-67 labeling index and Her2 amplification were also evaluated. Six of the 74 patients showed metastases at the diagnosis, and 8 patients developed metastases during the follow up. The median follow up time for the patients cohort was 44 months (range 28-57+). All metastatic patients (n=14) died from the disease. Genomic DNA extracted from 6 normal breast tissues obtained by reductive mammoplasty, and tumour samples was subjected to bisulphite treatment and the converted DNA was used as a template for MSP using primers specific for the methylated hsa-miR9 sequence. Results: Methylation at the hsa-miR9 promoter region was detected in 33 of 74 (44%) breast tumours and none of the 6 normal breast tissues (p=0.02). Interestingly, hsa-miR9 methylation was significantly more frequent in patients with syncronous or metachronous distant metastases (8 of 14, 57%) as compared with patients free from metastases (11 of 33, 32%) (p=0.02 χ2 Test). In particular, methylation was detected in all 5 tumours showing only bone metastases (100%), whereas methylation was less frequent (33%) in the group characterized by the the presence of visceral metastases (P=0.03 χ2 Test). Conclusions: Our results suggest that hsa-miR9 methylation in breast cancer is associated with tumour metastatic behaviour and might represent a novel biomarker for monitoring breast cancer patients.