Functional Screening of Candidate Micrornas in Hs-5 Stromal Cells Using a Jurkat Nf-κb-egfp Reporter Coculture Model
Year: 2026
Type: article
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.
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: diamond
OpenAlex ID: W7223241665