suggesting that re expression of these miR NAs is a result of a true epigenetic references alteration in the cells. We utilized the micro array platform to see which other chromosome 14 miRNAs could be induced using the combination of HDAC inhibitors and de methylating agents. Interestingly, out of all 65 chromosome 14 miRNAs assessed in four mel anoma cell lines, only five miRNAs were shown to be induced in any of the cell lines mir 127 3p, mir 137, mir 376a, mir 376c and mir 485 3p. These five miRNAs, expressed in normal melanocytes, could not be further up regulated in these cells in response to epigenetic modifiers. Four of these five miRNAs were found to be down regulated but not entirely silenced in nevi Inhibitors,Modulators,Libraries and melanoma.

Results obtained with the more sensitive method of qRT PCR verified that mir 376a, Inhibitors,Modulators,Libraries mir 376c and mir 136 can be significantly induced following treatment with epigenetic modifiers in most Inhibitors,Modulators,Libraries of the melanoma cell lines. Mir 127 was previously shown to target BCL 6 in a bladder cancer model, so we first generated melan oma cell lines that ectopically express mir 127 in a stable manner. In our experimental system, mir 127 over expression did not lead to a significant decrease in BCL 6 levels in melanoma cell lines, nor did it lead to a signifi cant change in melanoma cell line proliferation or migra tion in vitro. We therefore decided to focus on other miRNAs whose expression was shown to be down regulated but not entirely absent in melanoma and as a first step generated melanoma cell lines that ecto pically express either mir 376a or mir 376c.

Cells over expressing either mir 376a or mir 376c exhib ited attenuated growth relative to pTER transfected control cells. This effect was modest yet statistically significant, leading to approximately 25 30% decrease in cell growth after 96 hours. Inhibitors,Modulators,Libraries This growth pattern was also observed using a micro electronic biosensor system that allows real time monitoring of cell growth in vitro. Cellu lar migration was monitored using an in vitro transwell system. Mir 376a and mir 376c transfected cells showed significantly attenuated migration through a transwell membrane 24 hours after seeding relative to pTER transfected control cells. Migration was also monitored using the real time cell analyzer, this time asses sing cell density following passage through a membrane as described in.

Whereas pTER transfected control mel anoma cells Inhibitors,Modulators,Libraries exhibited a time dependent migration through the membrane, the mir 376a and mir 376c transfected enough cells showed almost no migration through the membrane within a 24 h period. Bioinformatic analysis using several web based tools showed that miRNA 376a and miRNA 376c have puta tive binding sites at the 3UTR of IGF1R, a tyrosine kinase receptor long known to be implicated in melanoma tumorigenesis and progression. The pu tative binding site of mir 376c is classified as 7mer 8mer binding, and that of mir 376a is classified as 8mer binding.