I. attribute to miR-145-5p and its direct targets pivotal roles in malignancy progression and in metastasis. = 6) collected from autopsies of patients deceased from causes other than lung cancer were used as Ropinirole control samples. Rabbit Polyclonal to MRPS24 In addition, to gain specificity we used brain metastases of melanoma or breast origin. Another comparator we utilized was normal lung tissue, collected from the peri-tumoral area of matched primary NSCLC lesions. These analyses uncovered consistent down-regulation of miR-145-5p expression in brain metastases, which we found to be caused by increased methylation of CpG islands in the 5 regulatory region of miR-145-5p. As a consequence, the abundance of OCT-4 and EGFR, two validated targets of miR-145-5p, was increased in primary lung cancer and their matched-brain metastasis compared to non-tumoral tissues. Treatment of lung cancer cells with inhibitors of DNA methylation, such as 5-azacytidine and vorinostat, restored miR-145-5p levels and concomitantly reduced expression of oncoproteins encoded by miR-145-5p target mRNAs. Altogether these findings imply that miR-145-5p down-regulation enables up-regulation of a group of target proteins, whose coordinate activity contributes to brain metastasis. RESULTS Deregulated microRNA expression between primary lung cancers and brain metastases To explore the involvement of miRs in brain we collected FFPE (Formalin-Fixed, Paraffin-Embedded) samples from patients affected by one of the 3 main types of Ropinirole tumors exhibiting the highest incidence of brain metastases, namely melanoma, breast and lung cancer. [3, 4]. In particular, we focused on 13 primary lung cancers and their matched brain metastases; for 10 of these 13 samples we disposed also of the normal lung tissues. In addition, we collected 16 unmatched lung-derived brain metastases. Our collection also included 6 brain metastases from melanoma, 9 brain metastases from breast cancer and 6 non-tumoral brain tissues derived from autopsy (Table ?(Table1).1). We profiled the expression of 906 human miRs in 13 primary lung cancers and their matched brain metastases, and 2 non-tumoral brain tissues. These analyses identified 8 miRs that were differentially expressed between primary lung tumors and brain Ropinirole metastases (Figure ?(Figure1A1A and Table Ropinirole ?Table2).2). In particular, 6 miRs (miR-219-2-3p, miR-219-5p, miR-124, miR-9*, miR-128, miR-338-3p) were up-regulated, while miR-145-5p and miR-1280 were down-regulated in brain metastases compared to primary lung cancers. Unsupervised principal component analysis (PCA) showed that the expression levels of these 8 miRs discriminated the group of primary lung cancer samples from that of brain metastases (Figure ?(Figure1B).1B). The significance level of the difference between signal distributions of the eight selected miRs within the 26 analyzed samples was determined with supervised statistical test (Supplementary Figure S1A). To further evaluate the reliability of these results, we analyzed the expression levels of one up-regulated miR, miR-219-5p, and one down-regulated miR, miR-145-5p, in all 13 matched samples (primary lung cancer and matched brain metastases) by qRT-PCR Ropinirole (Supplementary Figure S1B). These experiments confirmed the results obtained by the array analysis. Open in a separate window Figure 1 miR-145-5p expression is down-regulated in brain metastasesA. Heat map of the identified signature of 8 miRs differentially expressed in 13 brain metastasis (BM) versus 13 matched primary lung cancer (PLC). B. Unsupervised principal component analysis (PCA). C. qRT-PCR analysis of miR-145-5p expression levels in 10 normal lung (NL), 13 matched primary lung cancer (PLC), and 29 brain metastases from lung (13 matched and 16 unmatched) (BML). D. qRT-PCR analysis of miR-145-5p expression levels in 6 normal brain (NB), 29 brain metastases from lung (BML) and 15 brain metastases from melanoma (6) and breast (9) (BM). E. Schematic representation of miR-145-5p CpG island genomic localization. F.-G. Pyrosequencing analysis of miR-145-5p CpG island methylation status in a representative patient of the casuistry (NL= normal lung; PLC= primitive lung cancer; BM= brain metastases; p= patient; reg1= region 1; reg2= region 2). Table 1 Casuistry description the antitumoral effects of miR-145-5p, we subcutaneously injected nu/nu athymic mice with either human lung cancer cells, either control A549 cells, or A549 cells expressing an exogenous miR-145-5p (A549/miR-145-5p). We firstly observed that ectopic expression of miR-145-5p markedly reduced migration of A549 cells but had no effect on cell proliferation and viability (Supplementary Figure S6A-C). We found that the engraftment of A549/miR-145-5p cells was significantly less efficient than that of A549 control lung cancer cells (Figure ?(Figure2A).2A). To investigate the role of miR-145-5p on brain metastasis we performed intracranial orthotopically injection of.