BackgroundFor 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.MethodsBioinformatic 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.ResultsNorthern 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.ConclusionCollectively, 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.

Background: Advanced stage laryngeal squamous cell carcinoma (LSCC) has sustained one of the lowest five-year survival rates across all other head and neck squamous cancers. For several decades, Black patients have maintained a greater likelihood of presenting with advanced LSCC and a higher overall mortality rate compared with White patients. Even when accounting for sociodemographics, a disparity remains, underscoring the importance of investigating the biologic basis of this disparity. Differential microRNA (miRNA) expression has been linked to racially disparate clinical outcomes in other cancers including breast and prostate, but remains underdetermined in LSCC. We present our findings where we investigated miR-9 levels and their influence on LSCC tumorigenesis and chemoresistance using in vitro cell lines derived from Black and White patients. Methods: We previously identified miR-9-5p as significantly lower (5X) in Black compared with White advanced stage LSCC in vivo samples. For functional studies, we obtained two LSCC cell lines derived from a Black patient (UM-SCC-12) and from a White patient (UM-SCC-10A) (provided by Tom Carey, University of Michigan). After transfection with either miR-9 mimic or inhibitor, we used the scratch wound assay to assess cell migration and the cell titer blue assay to assess cell proliferation and chemoresistance. Results: Prior to transfection, we found that UM-SCC-12 had baseline increases in cellular migration, proliferation and chemoresistance compared to UM-SCC-10A. By Northern blot analysis, we further determined that mature miR-9-5p was inherently 5X lower in UM-SCC-12 compared with UM-SCC-10A. After transient transfection, we noted that overexpressing miR-9 in UM-SCC-12 resulted in decreased cellular migration, decreased proliferation and increased chemosensitivity compared to a mock oligo control, whereas inhibiting miR-9 in UM-SCC-10A resulted in increased cellular migration, increased proliferation and decreased chemosensitivity compared to a mock oligo control. Conclusion: Collectively, these studies support that low miR-9 may play a role in LSCC tumorigenesis and chemoresistance. In this manner, it may contribute to cancer health disparate outcomes observed in advanced stage LSCC. Future studies will involve characterizing significant downstream mediators of low miR-9-5p gene regulation. Citation Format: Christina Gobin, Samuel Inkabi, Chayil Lattimore, James Menefee, Tengfei Bian, Christopher Fields, Mingyi Xie, Chengguo Xing, Kristianna Fredenburg. Low miR-9 impacts tumorigenesis and chemoresistance in Black compared with White laryngeal squamous cell carcinoma [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C042.

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.