School of Medicine and Health Sciences Poster Presentations

Title

miR-p14, a Novel Tumor Suppressor microRNA, May Serve as a Therapeutic Target in Melanoma

Document Type

Poster

Abstract Category

Cancer/Oncology

Keywords

miR-p14, melanoma, biomarker, tumor suppressor

Publication Date

Spring 5-1-2019

Abstract

Melanoma remains an aggressive cancer, marked by an increasingly high rate of malignancy that accounts for more than 70% of skin-related deaths with a low five-year survival rate (5%) in advanced stages. Given a high predilection for early metastasis, frequent silencing of tumor suppressor gene p14ARF has been implicated as an important event in melanoma progression. However, the mechanism of p14ARF inactivation remains unclear. We previously characterized a deleted AT rich repeat (ATRR) sequence that encodes miR-p14, a novel microRNA (miRNA) positively correlated with p14ARF expression. Since dysregulated miRNAs have been demonstrated to modulate important hallmarks of cancer, the primary aim of this study was to identify potential target genes of miR-p14 and to delineate their roles in the course of melanoma tumorigenesis. Bioinformatic algorithms revealed high homology between miR-p14 and three bona fide miRNAs (miR-1277, miR-620, & miR-3171). Potential target genes of miR-p14 were identified using cross-referencing analysis across three microRNA databases. A well characterized malignant melanoma cell line, A375-SM, was obtained from ATCC and cultured in DMEM medium with 10% fetal bovine serum. miR-p14 mimic was transfected via Lipofectamine RNAiMAX to assess its effects on target gene expression. Total RNA was isolated and the efficacy of transfection was confirmed with TaqMan quantitative RT-PCR. Primers for candidate genes were designed using Primer-BLAST software. The expression of individual genes was assayed using qRT-PCR and further functional analysis of miR-p14 was performed by MTT assay. Cross-referencing analysis between three homologous microRNAs to miR-p14 revealed 10 predicted target genes in addition to p14ARF. Forced expression of miR-p14 resulted in the upregulation of two known tumor suppressors involved in the cell cycle and cell survival: p14ARF, an inducer of p53-dependent and p53-independent pathways, and PTEN, a negative regulator of the Akt/PKB signaling pathway. miR-p14 overexpression also upregulated SAP130, a damage-associated molecular pattern (DAMP) associated with necroptosis. MTT assay results further showed forced expression of miR-p14 inhibits cell proliferation. Taken together, our data supports the predicted role of novel miR-p14 to function as a tumor suppressor regulating p14ARF expression through a p53-independent mechanism in addition to other gene targets associated with an anti-tumor response. These findings suggest miR-p14 may serve as a potential therapeutic biomarker in melanoma management.

Open Access

1

Comments

Presented at Research Days 2019.

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miR-p14, a Novel Tumor Suppressor microRNA, May Serve as a Therapeutic Target in Melanoma

Melanoma remains an aggressive cancer, marked by an increasingly high rate of malignancy that accounts for more than 70% of skin-related deaths with a low five-year survival rate (5%) in advanced stages. Given a high predilection for early metastasis, frequent silencing of tumor suppressor gene p14ARF has been implicated as an important event in melanoma progression. However, the mechanism of p14ARF inactivation remains unclear. We previously characterized a deleted AT rich repeat (ATRR) sequence that encodes miR-p14, a novel microRNA (miRNA) positively correlated with p14ARF expression. Since dysregulated miRNAs have been demonstrated to modulate important hallmarks of cancer, the primary aim of this study was to identify potential target genes of miR-p14 and to delineate their roles in the course of melanoma tumorigenesis. Bioinformatic algorithms revealed high homology between miR-p14 and three bona fide miRNAs (miR-1277, miR-620, & miR-3171). Potential target genes of miR-p14 were identified using cross-referencing analysis across three microRNA databases. A well characterized malignant melanoma cell line, A375-SM, was obtained from ATCC and cultured in DMEM medium with 10% fetal bovine serum. miR-p14 mimic was transfected via Lipofectamine RNAiMAX to assess its effects on target gene expression. Total RNA was isolated and the efficacy of transfection was confirmed with TaqMan quantitative RT-PCR. Primers for candidate genes were designed using Primer-BLAST software. The expression of individual genes was assayed using qRT-PCR and further functional analysis of miR-p14 was performed by MTT assay. Cross-referencing analysis between three homologous microRNAs to miR-p14 revealed 10 predicted target genes in addition to p14ARF. Forced expression of miR-p14 resulted in the upregulation of two known tumor suppressors involved in the cell cycle and cell survival: p14ARF, an inducer of p53-dependent and p53-independent pathways, and PTEN, a negative regulator of the Akt/PKB signaling pathway. miR-p14 overexpression also upregulated SAP130, a damage-associated molecular pattern (DAMP) associated with necroptosis. MTT assay results further showed forced expression of miR-p14 inhibits cell proliferation. Taken together, our data supports the predicted role of novel miR-p14 to function as a tumor suppressor regulating p14ARF expression through a p53-independent mechanism in addition to other gene targets associated with an anti-tumor response. These findings suggest miR-p14 may serve as a potential therapeutic biomarker in melanoma management.