Cancer Res Treat.  2017 Oct;49(4):947-959. 10.4143/crt.2016.280.

Upregulation of MicroRNA-1246 Is Associated with BRAF Inhibitor Resistance in Melanoma Cells with Mutant BRAF

• Kim JH
• Ahn JH
• Lee M

• KMID: 2394814
• DOI: http://doi.org/10.4143/crt.2016.280

Abstract

PURPOSE
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.
MATERIALS AND 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.
CONCLUSION
These results suggest that miR-1246 may be a potential therapeutic target in melanoma with acquired resistance to BRAF inhibitors.

Keyword

MicroRNAs; BRAF; PLX 4720; Drug resistance; Melanoma; Microarray analysis

MeSH Terms

Apoptosis
Autophagy
Cell Cycle
Cell Death
Cell Line
Drug Resistance
Immunoblotting
Melanoma*
Microarray Analysis
MicroRNAs
Necrosis
Polymerase Chain Reaction
United Nations
Up-Regulation*
MicroRNAs

Figure

• 

  Fig. 1. Validation of miRNA microarray data using miRNA-specific real-time quantitative reverse-transcription polymerase chain reaction. Real-time reverse-transcription polymerase chain reaction was conducted to measure the mRNA expression levels of miR-17-3-p, miR-92a-1, miR-3617, miR-1246, and miR-193b-3p. The mean threshold cycle (Ct) was determined based on triplicate reactions. The 2−ΔΔCt method was used to calculate the fold differences in miRNA expression among the tested samples. The expression of the target genes was normalized to GAPDH expression. **p < 0.01 according to Dunnett’s t test in relative to BRAF inhibitor–sensitive A375P cells.

• 

  Fig. 2. MicroRNA-1246 controls the resistance to the BRAF inhibitor in melanoma cells. A375P (A), A375P/Mdr (B), and SK-MEL-2 (C) cells were transiently transfected with a miR-1246 mimic or inhibitor for 24 hours and then treated with PLX4720 in 96-well plates for 3 days. Cell growth was then evaluated using a MTT assay. The data represent the means (standard deviation) of quadruplicates from one of three representative experiments (A, C) or of three independent experiments (B). **p < 0.01 and *p < 0.05 compared with the negative control, according to Dunnett’s t test.

• 

  Fig. 3. Transfection with miR-1246 leads to decreased cell growth and G2/M arrest. A375P/Mdr cells were transiently transfected with miR-1246 mimic or control RNA for 24 hours, then treated with PLX4720 for 48 hours (A, B) or 72 hours (C). (A) Cell cycle progression was assessed by staining fixed cells with propidium iodide and by flow cytometric analysis (with proper preparation of the cells). The vertical and horizontal axes represent the cell number and DNA fluorescence intensity, respectively. The percentages of cells in individual cell cycle phases were quantified using the Cell Quest Pro software. The presented results are representative of at least three independent experiments. (B) Whole cell extracts were prepared at 48 hours after PLX4720 treatment. The expression levels of p21Cip1 and p27Kip1 were assessed by immunoblotting. β-Actin was assessed as a control for protein loading. The results presented are representative of two independent experiments. (C) Apoptosis was evaluated by staining with annexin V 72 hours after transfection. The flow cytometric profile shows the annexin V–FITC staining on the X-axis and the propidium iodide (PI) staining on the Y-axis. The numbers represent the percentage of cells in each quadrant. The presented results are representative of at least three independent experiments.

• 

  Fig. 4. Impaired autophagic flux during PLX4720-induced growth inhibition of A375P/Mdr cells. After transient transfection with a miR-1246 mimic or control RNA for 24 hours, autophagic flux was monitored in cells exposed to 20 μM PLX4720 for 48 hours. Autophagy was then detected by flow cytometric analysis of cells incubated with Cyto-ID green fluorescent probes, which detect autophagic vacuoles. (A) A representative histogram of the flow cytometric analysis is shown. Data are representative of at least two independent experiments. (B) The values are expressed as a fold increase, where the value observed in control RNA–transfected cells was set to 1.0. The upper panel shows a representative histogram of flow cytometric analysis. The lower panel shows the summarized data describing the percentage of cells positive for Cyto-ID fluorescence (n=3). CTL, control. *p < 0.05 compared with the vehicle control, as determined by Dunnett’s t test

• 

  Fig. 5. Effect of miR-1246 on MEK-ERK signaling. A375P/Mdr cells were transiently transfected with a miR-1246 mimic or control RNA for 24 hours. Cell lysates were then prepared after treatment with the indicated concentrations of PLX4720 for 24 hours. The phosphorylated forms of MEK and ERK were detected by immunoblotting using anti–p-MEK and anti–p-ERK antibodies. The same blots were stripped and reprobed with anti-MEK and anti-ERK antibodies to confirm similar expression levels of MEK and ERK proteins in all lanes. The numbers listed below each band indicate the phosphorylated protein/total protein ratios determined using the Image Lab software. Data are representative of at least three independent experiments.

Reference

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