Effect of microRNA-206 on G1 arrest by inhibition of CDK4, cyclin C, and cyclin D in melanoma cells.

Abstract

e20047 Background: MiR-206 has been implicated in a large number of cancers. However, its role in tumor biology is unknown and its biological function has yet to be fully characterized. To examine the role of miR-206 in cancer, we examined the expression of miR-206 in melanoma and identified potential target transcriptss that could be important for the progression of this disease. Methods: Using quantitative RT-PCR we compared expression of 364 microRNAs in melanoma skin biopsies skin from normal donors, melanoma cell lines, and normal melanocytes. The effects of miR-206 on cell growth, apoptosis, and cellular migration/invasion were determined using in vitro assays comparing melanoma cell lines to normal melanocytes. Putative mRNA targets of miR-206 were bioinfomatically identified, and empirically tested by luciferase-3’UTR reporter assays. The effect of miR-206 on the cell cycle of melanoma cells was assayed by flow cytometry. Results: Expression profiling of microRNAs in melanoma lesional skin biopsies compared to normal donor skin biopsies revealed numerous differentially regulated miRs. One such microRNA, miR-206, was significantly highly down-regulated in melanoma biopsies (-75.4-fold, p=1.7x10-4) compared to normal skin and normal melanocytes. Functional analysis showed that miR-206 substantially reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatic analysis identified the cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate mRNA targets of miR-206. Luciferase reporter gene assays revealed that miR-206 inhibits translation of CDK4, Cyclin D1, and Cyclin C. Consistent with this inhibition of CDK4 and Cyclin D1, miR-206 transfection induced robust G1 arrest in multiple melanoma cell lines. Conclusions: MiR-206 expression was decreased in melanoma tissue and cell lines compared to normal skin and melanocytes, respectively. Inhibition of Cyclin C, Cyclin D1 and CDK4 by miR-206 highlights its role in regulating cell cycle progression, a key aspect of melanoma progression. These observations support miR-206 as a potential tumor suppressor in melanoma, and possibly other cancers.