Downregulation of miR-4772-3p Promotes Enhanced Regulatory T Cell Capacity in Malignant Pleural Effusion by Elevating Helios Levels

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Downregulation of miR-4772-3p Promotes Enhanced Regulatory T Cell Capacity in Malignant Pleural Effusion by Elevating Helios Levels

Malignant pleural effusion (MPE) is a common and life-limiting complication of advanced malignancies, particularly non-small cell lung cancer (NSCLC). Its pathogenesis is closely linked to an immunosuppressive microenvironment dominated by regulatory T cells (Tregs). These Tregs suppress antitumor immunity, enabling tumor progression. Recent research has highlighted the critical role of the transcription factor Helios (IKAROS Family Zinc Finger 2, IKZF2) in stabilizing Treg functionality. This study investigates the molecular mechanism by which the microRNA miR-4772-3p regulates Helios expression in Tregs within MPE, offering insights into potential therapeutic strategies.

Immune Dysregulation in MPE: Tregs and Helios

Tregs, characterized by CD4, CD25, and FOXP3 expression, are pivotal in maintaining immune tolerance. However, their excessive accumulation in tumors correlates with poor prognosis. Helios, a transcription factor co-expressed with FOXP3, enhances Treg stability and suppressive capacity. This study analyzed Treg populations in MPE and peripheral blood mononuclear cells (PBMCs) from NSCLC patients, revealing a significant increase in Helios+ Tregs compared to non-malignant pleural effusions (NPE) and healthy controls. In PBMCs of NSCLC patients, Helios+ Tregs constituted 52.51% ± 5.80% of CD4+CD25+FOXP3+ Tregs, compared to 33.47% ± 4.37% in healthy individuals (P < 0.01). The proportion of Tregs in MPE (4.83% ± 0.43%) surpassed both PBMCs (4.16% ± 0.25%, P < 0.01) and NPE (3.51% ± 0.65%, P < 0.01), with Helios+ Tregs accounting for 2.96% ± 0.35% of CD4+ T cells in MPE versus 1.18% ± 0.38% in NPE (P < 0.01).

miRNA Profiling Identifies miR-4772-3p as a Key Regulator

Small RNA sequencing of mononuclear cells from MPE and NPE identified miR-4772-3p as significantly downregulated in MPE (P 2, P < 0.05) revealed miR-4772-3p levels inversely correlated with Helios protein (r = –0.631, P < 0.001) and mRNA (r = –0.468, P = 0.009) in MPE. Bioinformatic predictions using TargetScan, miRWalk, and DIANA-microT identified IKZF2 (Helios) as a putative target, with a conserved binding site in its 3’-untranslated region (3’-UTR).

Functional Validation of miR-4772-3p and Helios Interaction

Dual-luciferase reporter assays confirmed miR-4772-3p directly binds the 3’-UTR of IKZF2. HEK293T cells co-transfected with wild-type IKZF2 3’-UTR plasmids and miR-4772-3p mimics exhibited a 55% reduction in luciferase activity (P < 0.01). Mutating the binding site abrogated this effect, validating specificity.

In vitro experiments using induced Tregs (iTregs) generated from healthy donor CD4+CD25− T cells demonstrated miR-4772-3p’s regulatory role. Transfection with miR-4772-3p mimics reduced Helios protein expression by 37% (P < 0.01), while inhibitors increased it by 38% (P < 0.05). IKZF2 mRNA levels mirrored these changes, decreasing by 50% with mimics (P < 0.001) and rising with inhibitors. Ectopic overexpression of Helios via pcDNA3.1-Helios plasmids reversed mimic-induced suppression, restoring Helios expression to baseline levels (P < 0.01).

Clinical Implications: miR-4772-3p as a Therapeutic Target

The downregulation of miR-4772-3p in MPE creates a permissive environment for tumor immune evasion by enhancing Helios+ Treg activity. Restoring miR-4772-3p expression could destabilize Tregs, reducing their immunosuppressive function. This study provides mechanistic evidence that miR-4772-3p targets IKZF2 to regulate Treg activity, positioning it as a potential therapeutic target.

Experimental Methodology

  1. Sample Collection: Pleural effusions and peripheral blood were collected from 30 NSCLC patients (MPE), 30 NPE patients (19 parapneumonic, 11 tuberculous), and 20 healthy controls. Mononuclear cells were isolated via Ficoll-Hypaque gradient centrifugation.
  2. Flow Cytometry: Tregs (CD4+CD25+FOXP3+) and Helios expression were quantified using fluorescent antibodies (BD Biosciences). Gating strategies excluded non-viable cells, with FOXP3 and Helios detected via intracellular staining.
  3. RNA Sequencing: Total RNA from six PEMC samples underwent sequencing (Illumina HiSeq X-ten). Differentially expressed miRNAs were analyzed using DESeq2.
  4. qRT-PCR: miR-4772-3p and IKZF2 expression were normalized to U6 and GAPDH, respectively. Primer sequences were validated for specificity.
  5. In Vitro Treg Induction: CD4+CD25− T cells from healthy donors were polarized into iTregs using TGF-β1 (5 ng/mL), anti-CD3/CD28 stimulation, and IL-2 (2 ng/mL) over seven days.
  6. Luciferase Assays: HEK293T cells were transfected with wild-type or mutant IKZF2 3’-UTR plasmids and miRNA mimics/inhibitors. Luciferase activity was measured 24 hours post-transfection.

Key Findings and Significance

  • Helios+ Treg Enrichment in MPE: NSCLC patients exhibited elevated Helios+ Tregs in MPE and PBMCs, correlating with disease progression.
  • miR-4772-3p Dysregulation: Reduced miR-4772-3p levels in MPE inversely correlated with Treg frequency and Helios expression, establishing a direct regulatory link.
  • Mechanistic Pathway: miR-4772-3p targets IKZF2, modulating Treg stability and suppressive function.
  • Therapeutic Potential: Restoring miR-4772-3p expression could destabilize Helios+ Tregs, potentially reversing immune suppression in MPE.

Conclusion

This study elucidates a novel mechanism by which miR-4772-3p deficiency in MPE upregulates Helios, enhancing Treg-mediated immunosuppression. Targeting this axis may offer new strategies for MPE treatment, particularly in NSCLC. Future research should explore in vivo miRNA delivery systems and combination therapies with immune checkpoint inhibitors.

doi.org/10.1097/CM9.0000000000000517

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