RESUMO
Background: For several decades, Black patients have carried a higher burden of laryngeal cancer among all races. Even when accounting for sociodemographics, a disparity remains. Differentially expressed microRNAs have been linked to racially disparate clinical outcomes in breast and prostate cancers, yet an association in laryngeal cancer has not been addressed. In this study, we present our computational analysis of differentially expressed miRNAs in Black compared with White laryngeal cancer and further validate microRNA-9-5p (miR-9-5p) as a potential mediator of cancer phenotype and chemoresistance. Methods: Bioinformatic analysis of 111 (92 Whites, 19 Black) laryngeal squamous cell carcinoma (LSCC) specimens from the TCGA revealed miRNAs were significantly differentially expressed in Black compared with White LSCC. We focused on miR-9-5 p which had a significant 4-fold lower expression in Black compared with White LSCC (p<0.05). After transient transfection with either miR-9 mimic or inhibitor in cell lines derived from Black (UM-SCC-12) or White LSCC patients (UM-SCC-10A), cellular migration and cell proliferation was assessed. Alterations in cisplatin sensitivity was evaluated in transient transfected cells via IC50 analysis. qPCR was performed on transfected cells to evaluate miR-9 targets and chemoresistance predictors, ABCC1 and MAP1B. Results: Northern blot analysis revealed mature miR-9-5p was inherently lower in cell line UM-SCC-12 compared with UM-SCC-10A. UM -SCC-12 had baseline increase in cellular migration (p < 0.01), proliferation (p < 0.0001) and chemosensitivity (p < 0.01) compared to UM-SCC-10A. Increasing miR-9 in UM-SCC-12 cells resulted in decreased cellular migration (p < 0.05), decreased proliferation (p < 0.0001) and increased sensitivity to cisplatin (p < 0.001). Reducing miR-9 in UM-SCC-10A cells resulted in increased cellular migration (p < 0.05), increased proliferation (p < 0.05) and decreased sensitivity to cisplatin (p < 0.01). A significant inverse relationship in ABCC1 and MAP1B gene expression was observed when miR-9 levels were transiently elevated or reduced in either UM-SCC-12 or UM-SCC-10A cell lines, respectively, suggesting modulation by miR-9. Conclusion: Collectively, these studies introduce differential miRNA expression in LSCC cancer health disparities and propose a role for low miR-9-5p as a mediator in LSCC tumorigenesis and chemoresistance.
RESUMO
BACKGROUND/AIM: miRNA functional analysis involves transfection with miRNA-based oligos to identify gain-of or loss-of function cellular phenotypes. Apoptosis is a common phenotypic endpoint for miRNA functional analysis. We report that four common cell dissociation enzymes, TrypLE, Accutase, Trypsin, and Accumax, can differentially impact cell viability and apoptosis in Annexin V flow cytometric analysis after miRNA-based transient transfection. MATERIALS AND METHODS: We transiently transfected a nonsense oligo into an epithelial cancer cell line (UM-SCC-12) for 24 h. Cells were harvested with either TrypLE, Accutase, Accumax, or Trypsin after 5 min. The Annexin V/7-AAD assay via flow cytometry was employed. Studies were performed in triplicate. Significant effects were detected by ANOVA, followed by Tukey's Multiple Comparison tests. RESULTS: Trypsin produced the lowest cell viability and lowest percentage of apoptotic cells, specifically when compared to TrypLE and Accutase, respectively (p<0.01). Importantly, transfected trypsinized cells had a significant difference in cell viability and necrosis (p<0.05) when compared with non-transfected trypsinized cells, highlighting the influence of miRNA-based transfection on Annexin V flow cytometric outcomes. Interassay variability was lowest with TrypLE (1.13 %). As such, TrypLE provided the greatest reproducibility and reliability in our cell line. CONCLUSION: Our study highlights the variable effects of cell dissociation enzymes on transfected cells. Overall, the variability may lead to errors in detection of apoptotic cells using the Annexin V assay after miRNA-based transfection. Before assay use, we recommend pretesting cell dissociation enzymes on transfected cells to ensure reliable and reproducible results.
