DIFFERENTIAL REGULATION OF AGGRESSIVE FEATURES IN MELANOMA CELLS BY SAME CLUSTER MICRORNA-17 AND -20A

Eyal Greenberg ^1,2 Steven Hajdu ¹ Yael Nemlich ^1,2 Ronit Cohen ^1,2 Rona Ortenberg ^1,2 Orit Itzhaki ¹ Jasmine Jacob ³ Michal J. Besser ^1,2 Yona Keisari ² Jacob Schachter ¹ Gal Markel ^1,2,4

¹Ella Institute of Melanoma, Sheba Medical Center, Tel Hashomer
²Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv
³Cancer Research Center, Sheba Medical Center, Tel Hashomer
⁴Talpiot Medical Leadership Program, Sheba Medical Center, Tel Hashomer

Background: MicroRNAs (miRNAs) are short non-coding RNAs that regulate the transcriptome epigenetically, and affect various cancer related functions.

Objective: To study the impact of same cluster two miRNAs on aggressive melanoma phenotype, and delineate the underlying molecular mechanisms.

Methods: A comparative miRNA array was performed on two isogenic sub-lines, which differ in tumorigenicity and related characteristics. miR-20a and miR-17 (17/92 cluster) expression levels were validated with qPCR. miR-20a/17 were over-expressed in melanoma cells by transfection. Transfectants were analyzed functionally in proliferation. Target validation with both miRNAs was performed using a luciferase reporter assay.

Results: miR-20a and miR-17 expression was higher in the more aggressive cell line, and accordingly, in 11 primary melanoma cultures compared to melanocytes. Paradoxically, over-expression of miR-20a in melanoma cells inhibited net proliferation in-vitro. In contrast, over-expression of miR-17, which differs from miR-20a in only two nucleotides and has identical seed region and predicted targets as miR-20a, resulted in no significant change in proliferation. A differential regulation of five exemplar (ADAR1, ITGB8, TGFBR2, MMP2 and VEGFA) predicted targets of miR-17/20a was validated with qPCR in a melanoma cell line transfected with miR-20a vs. miR-17. Finally, a luciferase reporter assay on a validated exemplar miR-17 target (ADAR1) was conducted on both miR-17 and miR-20a mimetic transfected cells as compared to scramble sequence transfection. Remarkably, ADAR1 was ratified as miR-17 direct target, whereas, miR-20a failed to target this gene due to the two nucleotides variation outside the seed region.

Conclusions: Despite the fact that miR-17 and miR-20a arise from the same cluster and have identical seed regions, they confer opposite functional effects and target different genes. This phenomenon was explained here by two nucleotides variation located outside the seed region and suggests differential regulatory roles for these two same cluster miRNAs.