The role of miR-18b in MDM2-p53 pathway signaling and melanoma progression.
BACKGROUND
Although p53 is inactivated by point mutations in many tumors, melanomas infrequently harbor mutations in the p53 gene. Here we investigate the biological role of microRNA-18b (miR-18b) in melanoma by targeting the MDM2-p53 pathway.


METHODS
Expression of miR-18b was examined in nevi (n = 48) and melanoma (n = 92) samples and in melanoma cell lines and normal melanocytes. Immunoblotting was performed to determine the expression of various proteins regulated by miR-18b. The effects of miR-18b overexpression in melanoma cell lines were investigated using assays of colony formation, cell viability, migration, invasion, and cell cycle and in a xenograft model (n = 10 mice per group). Chromatin immunoprecipitation and methylation assays were performed to determine the mechanism of microRNA silencing.


RESULTS
Expression of miR-18b was substantially reduced in melanoma specimens and cell lines by virtue of hypermethylation and was reinduced (by 1.5- to 5.3-fold) in melanoma cell lines after 5-AZA-deoxycytidine treatment. MDM2 was identified as a target of miR-18b action, and overexpression of miR-18b in melanoma cells was accompanied by 75% reduced MDM2 expression and 2.5-fold upregulation of p53, resulting in 70% suppression of melanoma cell colony formation. The effects of miR-18b overexpression on the p53 pathway and on melanoma cell growth were reversed by MDM2 overexpression. Stable overexpression of miR-18b produced potent tumor suppressor activity, as evidenced by suppressed melanoma cell viability, induction of apoptosis, and reduced tumor growth in vivo. miR-18b overexpression suppressed melanoma cell migration and invasiveness and reversed epithelial-to-mesenchymal transition.


CONCLUSIONS
Our results demonstrate a novel role for miR-18b as a tumor suppressor in melanoma, identify the MDM2-p53 pathway as a target of miR-18b action, and suggest miR-18b overexpression as a novel strategy to reactivate the p53 pathway in human tumors.
Center for Melanoma Research and Treatment, California Pacific Medical Center Research Institute, 475 Brannan St, Ste 220, San Francisco, CA 94107, USA.
Altaf A
AA
Dar
Shahana
S
Majid
Claudia
C
Rittsteuer
David
D
de Semir
Vladimir
V
Bezrookove
Schuyler
S
Tong
Mehdi
M
Nosrati
Richard
R
Sagebiel
James R
JR
Miller
Mohammed
M
Kashani-Sabet
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002465:Cell Movement; D002470:Cell Survival; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D058750:Epithelial-Mesenchymal Transition; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D008156:Luciferases / Q000032:analysis; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D014411:Neoplastic Stem Cells; D017354:Point Mutation; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014183:Transplantation, Heterologous; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D000964:Antimetabolites, Antineoplastic; C540193:MIrn181 microRNA, human; D035683:MicroRNAs; D016159:Tumor Suppressor Protein p53; D008156:Luciferases; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D001374:Azacitidine