Overexpression of miR-3151 leads to direct deregulation of the TP53 pathway and is associated with BRAF mutations in malignant melanoma
Advisor:de la Chapelle, Albert
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Series/Report no.:2014 Richard J. and Martha D. Denman Undergraduate Research Forum. 19th
The BRAF gene is the most frequently mutated gene in malignant melanoma (MM). When mutated, it is associated with a more aggressive disease phenotype. MicroRNAs are small non-coding RNAs that downregulate the expression of their target genes by binding to their 3′-UTR. Recently, microRNA miR-3151 was identified in intron 1 of BAALC, the most upregulated gene in BRAF mutated melanoma. In acute myeloid leukemia, both high miR-3151 and high BAALC are associated with poor survival. In addition, miR-3151 has leukemogenic activity via direct deregulation of TP53. To begin to decipher miR-3151’s role in MM carcinogenesis, miR-3151 and TP53 expression levels were determined in MM patients (n=20). Indeed, patients with high miR-3151 had lower TP53 expression. To prove direct downregulation of TP53 by miR-3151, we stably expressed miR-3151 and antagomiR-3151 in MM cell lines. RT-PCR and Western blots confirmed that miR-3151 upregulated and antagomiR-3151 downregulated TP53. Increased levels of miR-3151 led to increased cell proliferation and decreased apoptosis (cell death), whereas antagomiR-3151 reduced cell growth and increased apoptosis. Next, we determined the BRAF mutation status and miR-3151 expression in the set of 20 MM patients. BRAF mutated (BRAFmut) patients had a 5-fold higher miR-3151 expression compared to BRAF wild-type (BRAFwt) patients. To test whether BRAF mutations directly influence miR-3151 expression, we silenced BRAF in BRAFmut cell lines (A375, Mel-39) and introduced the BRAF mutation into a BRAFwt cell line (MeWo). Silencing BRAF decreased miR-3151 expression and increased TP53 expression, while introducing the BRAF mutation increased miR-3151 expression and decreased TP53 when compared to scramble control. In conclusion, miR-3151 downregulated TP53 in MM cell lines, thereby increasing the cell proliferation and decreasing apoptosis. AntagomiR-3151 reduced MM cell growth and increased apoptosis. High miR-3151 expression is associated with the presence of BRAF mutations in MM patients. BRAFmut can directly increase miR-3151 expression.
Health Professions - Laboratory/Cellular: 3rd Place (The Ohio State University Denman Undergraduate Research Forum)
Academic Major: Molecular Genetics