FUNCTIONAL SCREENING OF CANDIDATE MICRORNAS IN HS-5 STROMAL CELLS USING A JURKAT NF-ΚB-EGFP REPORTER COCULTURE MODEL
- 1Fundação Hemocentro de Ribeirão Preto (FUNDHERP), Ribeirão Preto, SP, Brazil
Abstract
Background: The tumor microenvironment (TME) is a complex and dynamic ecosystem where stromal and immune cells interact to influence tumor progression and immune responses. Stromal cells are active regulators of this environment, capable of shaping immune cell behavior through the production of soluble factors, extracellular matrix components and immunomodulatory signals. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level and can reprogram cellular phenotypes and intercellular communication. Although extensively studied in tumor cells, the functional contribution of miRNAs within stromal cells and their impact on immune activation remain poorly understood. Objectives: Therefore, this study aimed to functionally evaluate candidate miRNAs introduced into HS-5 stromal cells and determine their effects on NF-κB activation in cocultured Jurkat reporter T cells. Methods: HS-5 stromal cells were reverse-transfected with individual pre-miRNAs (50 nM). After 48h, PMA/ionomycin-preactivated Jurkat NF-κB-eGFP reporter cells were added to establish stromal-immune cocultures. High-content screening (HCS) images were acquired immediately after coculture establishment (0 h) and after 6, 12 and 24h. Automated image analysis enabled discrimination of cell populations and quantification of Jurkat NF-κB activation through two independent parameters: the percentage of eGFP-positive cells and mean fluorescence intensity (MFI). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test. Results: All experimental conditions displayed similar activation kinetics, with high initial eGFP expression following PMA/ionomycin stimulation, a transient increase at 6h and progressive reduction at 24h. Differences among miRNA-treated groups were limited during early time points but became more evident after 24h, indicating that stromal miRNA modulation predominantly affected the maintenance of NF-κB activation rather than the initial activation response. Eight candidate miRNAs significantly reduced Jurkat NF-κB activation compared with the negative control (p<0.05): miR-17, miR-19b, miR-20a, miR-22, miR-30a, miR-145, miR-181d and miR-222. Notably, miR-17, miR-19b and miR-20a, which belong to the miR-17-92 cluster, were among the identified candidates, suggesting a potential role of this regulatory network in stromal-mediated immune modulation. Importantly, the percentage of eGFP-positive cells and MFI showed highly concordant patterns across conditions, supporting the reliability of the HCS-based functional assessment. Conclusions: This functional screening identified candidate miRNAs capable of modulating the ability of HS-5 stromal cells to sustain NF-κB activation in Jurkat cells. The enrichment of miR-17-92 cluster members among the significant candidates highlights potential miRNA-driven mechanisms involved in stromal-immune communication within the tumor microenvironment. These findings provide a robust foundation for prioritizing candidate miRNAs for future mechanistic validation and large-scale functional screening approaches.
Related
- Reporter Virus-Based High-Throughput Screening Identifies LM-10 as a Novel Inhibitor Targeting KRM1 for Coxsackievirus A10 Blockade
- Exploratory Analysis of Selected MicroRNAs as Tissue-Based Candidate Biomarkers in Ovarian Cancer
- Circulating microRNAs as potential candidate biomarkers of left ventricular reverse remodeling after myocardial infarction: A pilot study
- Reframing liquid biopsy microRNAs as functional readouts of Tumor–Microenvironment cross-talk
- Screening traditional Chinese medicine monomers for premature ovarian insufficiency: from network meta-analysis to the effects of a promising candidate on oxidative stress biomarkers
BibTeX
@article{silva2026functional,
title = {{FUNCTIONAL SCREENING OF CANDIDATE MICRORNAS IN HS-5 STROMAL CELLS USING A JURKAT NF-ΚB-EGFP REPORTER COCULTURE MODEL}},
author = {Larissa Ignácio SILVA and João Pedro Zanon MURAROLLI and Giovana Batriz Capuz MORETO and Rodrigo Alexandre PANEPUCCI},
journal = {Hematology, Transfusion and Cell Therapy},
year = {2026},
month = oct,
doi = {10.1016/j.htct.2026.108111},
url = {https://doi.org/10.1016/j.htct.2026.108111},
note = {CC BY},
}
RIS
TY - JOUR
TI - FUNCTIONAL SCREENING OF CANDIDATE MICRORNAS IN HS-5 STROMAL CELLS USING A JURKAT NF-ΚB-EGFP REPORTER COCULTURE MODEL
AU - Larissa Ignácio SILVA
AU - João Pedro Zanon MURAROLLI
AU - Giovana Batriz Capuz MORETO
AU - Rodrigo Alexandre PANEPUCCI
PY - 2026
DA - 2026/10/23
JO - Hematology, Transfusion and Cell Therapy
DO - 10.1016/j.htct.2026.108111
UR - https://doi.org/10.1016/j.htct.2026.108111
AB - Background: The tumor microenvironment (TME) is a complex and dynamic ecosystem where stromal and immune cells interact to influence tumor progression and immune responses. Stromal cells are active regulators of this environment, capable of shaping immune cell behavior through the production of soluble factors, extracellular matrix components and immunomodulatory signals. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level and can reprogram cellular phenotypes and intercellular communication. Although extensively studied in tumor cells, the functional contribution of miRNAs within stromal cells and their impact on immune activation remain poorly understood. Objectives: Therefore, this study aimed to functionally evaluate candidate miRNAs introduced into HS-5 stromal cells and determine their effects on NF-κB activation in cocultured Jurkat reporter T cells. Methods: HS-5 stromal cells were reverse-transfected with individual pre-miRNAs (50 nM). After 48h, PMA/ionomycin-preactivated Jurkat NF-κB-eGFP reporter cells were added to establish stromal-immune cocultures. High-content screening (HCS) images were acquired immediately after coculture establishment (0 h) and after 6, 12 and 24h. Automated image analysis enabled discrimination of cell populations and quantification of Jurkat NF-κB activation through two independent parameters: the percentage of eGFP-positive cells and mean fluorescence intensity (MFI). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test. Results: All experimental conditions displayed similar activation kinetics, with high initial eGFP expression following PMA/ionomycin stimulation, a transient increase at 6h and progressive reduction at 24h. Differences among miRNA-treated groups were limited during early time points but became more evident after 24h, indicating that stromal miRNA modulation predominantly affected the maintenance of NF-κB activation rather than the initial activation response. Eight candidate miRNAs significantly reduced Jurkat NF-κB activation compared with the negative control (p<0.05): miR-17, miR-19b, miR-20a, miR-22, miR-30a, miR-145, miR-181d and miR-222. Notably, miR-17, miR-19b and miR-20a, which belong to the miR-17-92 cluster, were among the identified candidates, suggesting a potential role of this regulatory network in stromal-mediated immune modulation. Importantly, the percentage of eGFP-positive cells and MFI showed highly concordant patterns across conditions, supporting the reliability of the HCS-based functional assessment. Conclusions: This functional screening identified candidate miRNAs capable of modulating the ability of HS-5 stromal cells to sustain NF-κB activation in Jurkat cells. The enrichment of miR-17-92 cluster members among the significant candidates highlights potential miRNA-driven mechanisms involved in stromal-immune communication within the tumor microenvironment. These findings provide a robust foundation for prioritizing candidate miRNAs for future mechanistic validation and large-scale functional screening approaches.
SN - 2531-1379
SN - 2531-1387
ER -