A Three-MicroRNA Panel in Serum as Novel Biomarker for Papillary Thyroid Carcinoma Diagnosis

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A Three-MicroRNA Panel in Serum as Novel Biomarker for Papillary Thyroid Carcinoma Diagnosis

Thyroid cancer, a leading malignancy of the endocrine system, is categorized into papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC). Among these, PTC accounts for 85%–90% of cases, highlighting its clinical significance. Despite advancements in diagnostic techniques like fine-needle aspiration biopsy (FNA) and imaging modalities, limitations such as invasiveness, cost, and operator dependency necessitate the development of non-invasive, reliable biomarkers. MicroRNAs (miRNAs), small non-coding RNAs regulating gene expression post-transcriptionally, have emerged as promising candidates due to their stability in circulation and dysregulation in cancers. This study identified a serum-based three-miRNA panel with diagnostic potential for PTC.

Study Design and Methodology

Conducted between 2015 and 2017, the research involved four stages: screening, training, testing, and external validation. Serum samples from 100 PTC patients, 96 healthy controls (HCs), and 30 nodular goiter (NG) patients were analyzed. Tissue samples from 23 PTC patients and matched adjacent normal tissues, along with serum-derived exosomes from 24 PTC patients and 24 HCs, were also examined.

In the screening stage, pooled serum samples (20 PTC, 20 NG, and 10 HCs) underwent profiling using the Exiqon miRNA qPCR panel, which evaluates 179 miRNAs. Forty differentially expressed miRNAs (36 upregulated, 4 downregulated) were identified using criteria: Ct 1.5 or <0.67. Four additional miRNAs (miR-95-5p, miR-190a-5p, miR-151a-5p, miR-222-3p) from literature were included, yielding 44 candidates.

Subsequent training (34 PTC vs. 35 HCs), testing (48 PTC vs. 45 HCs), and external validation (18 PTC vs. 16 HCs) stages utilized qRT-PCR. RNA extraction from serum or exosomes employed the mirVana PARIS Kit, with synthetic cel-miR-39 for normalization. Tissue RNA was isolated via Trizol. Reverse transcription and amplification used Bulge-Loop™ primers on a LightCycler® 480 system. Data analysis via the 2^-ΔΔCt method compared miRNA expression between groups.

Key Findings

Identification of Diagnostic miRNAs

Three miRNAs—miR-25-3p, miR-296-5p, and miR-92a-3p—were consistently upregulated in PTC serum across all stages:

  • miR-25-3p: Median FC = 1.542 (testing stage), P < 0.001.
  • miR-296-5p: Median FC = 2.017 (testing stage), P < 0.001.
  • miR-92a-3p: Median FC = 1.714 (testing stage), P < 0.001.

A logistic regression model combining these miRNAs generated a diagnostic panel:
Logit(P) = 1.768 – 0.009 × miR-25-3p – 0.187 × miR-296-5p – 0.797 × miR-92a-3p.

Diagnostic Performance

  • Training stage: AUC = 0.727 (95% CI: 0.600–0.855), sensitivity = 65.7%, specificity = 73.3%.
  • Testing stage: AUC = 0.771 (95% CI: 0.669–0.874), sensitivity = 88.9%, specificity = 68.9%.
  • External validation: AUC = 0.862 (95% CI: 0.734–0.990), sensitivity = 93.3%, specificity = 66.7%.
  • Combined analysis: AUC = 0.775 (95% CI: 0.707–0.843), outperforming individual miRNAs.

The panel excelled in differentiating PTC from NG (AUC = 0.969, 95% CI: 0.927–1.000), underscoring its specificity for malignancy.

Tissue and Exosome Analysis

Contrary to serum findings, miR-25-3p (P = 0.025) and miR-92a-3p (P = 0.043) were downregulated in PTC tissues, while miR-296-5p showed no significant change. TCGA data (59 PTC vs. normal tissues) corroborated reduced miR-25 and miR-296 expression. In serum-derived exosomes, miR-296-5p remained upregulated (P = 0.019), whereas miR-25-3p (P < 0.001) and miR-92a-3p (P = 0.006) were downregulated, suggesting distinct miRNA trafficking mechanisms.

Bioinformatics Insights

DIANA-TarBase v7.0 and miRPath v3.0 analyses linked the miRNAs to cancer-related pathways:

  • KEGG: Viral carcinogenesis, lysine degradation, cell cycle.
  • GO: Cell death, protein binding, metabolic processes.

Clinical Implications and Limitations

The three-miRNA panel offers a non-invasive, cost-effective tool for PTC diagnosis, particularly beneficial for equivocal FNA results. Its high AUC in differentiating PTC from NG reduces unnecessary surgeries. However, limitations include:

  1. Sample size: Larger cohorts are needed for validation.
  2. Mechanistic clarity: Roles of miRNAs in PTC pathogenesis require further exploration.
  3. Form-specific expression: Discrepancies between serum, tissue, and exosome miRNA levels warrant investigation into miRNA release mechanisms.

Conclusion

This study establishes miR-25-3p, miR-296-5p, and miR-92a-3p as a robust serum biomarker panel for PTC diagnosis. The combination’s high sensitivity and specificity, validated across multiple stages, positions it as a transformative tool in clinical practice. Future research should address mechanistic insights and expand validation to diverse populations and thyroid cancer subtypes.

doi.org/10.1097/CM9.0000000000001107

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