Abberant Regulation of Nerve Growth Factor Receptor (NGF-R) by micro-RNAs in Melanoma

1. B1 14PA Control 377 Control 377 NHEM 14PA B1 NGFR NGFR Actin Actin Abberant Regulation of Nerve Growth Factor Receptor (NGF-R) by micro-RNAs in Melanoma Raya Leibowitz-Amit1,4 , Hagit Schayek1, Liron Zehavi1,2, Yehezkel Sidi1,2,3, Raphael Catane1 and Dror Avni3 NHEM Nevi Melanoma NHEM Nevi Melanoma 1Laboratory of Molecular Cell Biology, Center for Cancer Research, Tel Hashomer, Israel. 2Department of molecular genetics and clinical biochemistry, Sackler School of Medicine, Tel Aviv University, Israel. 3Department of Medicine C, Chaim Sheba Medical Center, Tel Hashomer, Israel. 4Department of Oncology, Sheba Medical Center, Tel Hashomer, Israel. Introduction & Aim Stable expression of mir-377 leads to significant decrease in the level of both NGF-R mRNA and protein • Metastatic melanoma is a devastating disease with limited treatment options.  • MicroRNAs (miRNAs) are a large family of post-transcriptional regulators of gene expression that control many developmental and cellular processes and who have major roles in cancer development.  • Recently, the growth and metastasis of melanoma has been proposed to be driven by a small subpopulation of melanoma stem cells that can be distinguished from non-tumorigenic melanoma cells based on the expression of Nerve Growth factor Receptor (NGFR). • We previously showed that the expression level of many miRNAs from a large cluster on chromosome 14q32 is significantly decreased in melanoma. • We observed that the 3' UTR of the mRNA of NGFR contains potential binding sites for mir-377, one of the miRNAs from the large miRNA cluster on 14q32.   • The aim of our current research was to investigate whether mir-377 is a negative regulator of NGF-R. A. B. Negative correlation between the expression levels of NGFR and Mir-377 in melanoma cells and melanocytes (A)The expression level of mir-377 (right) and the mRNA of NGF-R (left) in 14PA (upper) and B1 (lower) melanoma cell lines following stable transfection with a control vector (HTR) or with mir-377. (B) The expression of the NGFR protein following transfection with control or with mir-377 vector. A. The 3' UTR of NGFR is a target of mir-377 B. 14PA-HTR/377 stable cells were transfected with the luciferase-NGFR-3'UTR plasmid or its empty control. After 48h, a standard luciferase reporter assay was performed. (A) The expression levels of mir-377 (right) and the mRNA of NGF-R (left) in normal melanocytes (NHEM) and in two melanoma cell lines as assessed by qRT-PCR. (B) The expression of the NGFR protein in the same cell lines as assessed by western blot. Actin serves as an internal loading control. Summary • NGFR is expressed in normal melanocytes but not in melanoma cell lines and samples. In contrast, mir-377 is expressed in normal melanocytes and in benign nevi but not in melanoma samples or in melanoma cell lines. • stable expression of mir-377 in melanoma cell lines led to a significant decrease in the level of both NGF-R mRNA and protein. Reporter assays using the luciferase gene attached to the 3'UTR of NGF-R showed that luciferase expression is decreased following over-expression of mir-377, proving that NGFR is a direct target of mir-377. • Our work points to the existence of a negative regulatory mechanism on NGFR by mir-377, not yet described until now. Moreover, our results may suggest that silencing of mir-377 may be involved in the pathogenesis of melanoma. Negative correlation between the expression levels of NGFR and Mir-377 in normal melanocytes, benign nevi and melanoma samples This work was supported by grants from the Israeli Cancer association (ICA), The ‘American Physician’s Fellowship’ (AFP), The Israeli Scientific Foundation (ISF), The Chief Scientist at the Israeli Ministry of Health and the Melanoma Research Alliance (MRA). R.L-A is a member of the ‘Talpiot medical leadership program’ at the Sheba Medical Center. The expression levels of mir-377 and the mRNA of NGFR in normal melanocytes, benign nevi and melanoma samples as assessed by qRT-PCR. Each diamond represents a sample. The square represents the median value