title: Upregulation of MicroRNA-1246 Is Associated with BRAF Inhibitor Resistance in Melanoma Cells with Mutant BRAF.
abstract: OBJECTIVE
  Intrinsic and acquired resistance limit the therapeutic benefits of inhibitors of oncogenic BRAF in melanoma. To identify microRNAs (miRNAs) associated with resistance to a BRAF inhibitor, we compared miRNA expression levels in three cell lines with different BRAF inhibitor sensitivity.
  
  
  METHODS
  miRNA microarray analysis was conducted to compare miRNA expression levels. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to confirm the expression of differentially expressed miRNAs. The cellular effects of miR-1246 were further examined by MTT assay, immunoblotting analysis, cell cycle analysis, flow cytometric assay of apoptosis, and autophagy assay.
  
  
  RESULTS
  The miRNA microarray analysis and qRT-PCR identified five miRNAs (miR-3617, miR-92a-1, miR-1246, miR-193b-3p, and miR-17-3p) with expression that was consistently altered in two BRAF inhibitor-resistant cell lines. Among the five miRNAs, a miR-1246 mimic significantly reduced the antiproliferative effects of the BRAF inhibitor PLX4720 in BRAF inhibitor-resistant A375P (A375P/Mdr) cells, suggesting that miR-1246 upregulation confers acquired resistance to BRAF inhibition. In particular, apoptosis was identified as a major type of cell death in miR-1246-transfected cells; however, necrosis predominated in mimic-control-transfected cells, indicating that the resistance to PLX4720 in miR-1246 mimic-transfected cells is predominantly due to a reduction in necrosis. Furthermore, we found that miR-1246 promoted G2/M arrest through autophagy as a way to escape cell death by necrosis and apoptosis in response to PLX4720. The promotion of BRAF inhibitor resistance by miR-1246 was associated with lowered levels of p-ERK.
  
  
  CONCLUSIONS
  These results suggest that miR-1246 may be a potential therapeutic target in melanoma with acquired resistance to BRAF inhibitors.
authors.affiliation: Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, Korea.
authors.forename: Jae-Hyeon
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Genome Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea.
authors.forename: Jun-Ho
authors.initials: JH
authors.lastname: Ahn
authors.affiliation: Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, Korea.
authors.forename: Michael
authors.initials: M
authors.lastname: Lee
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics*; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics*; D034622:RNA Interference; D015203:Reproducibility of Results
chemical_list: D000970:Antineoplastic Agents; C577700:MIRN1246 microRNA, human; D035683:MicroRNAs; D047428:Protein Kinase Inhibitors; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; Drug resistance; Melanoma; MicroRNAs; Microarray analysis; PLX 4720