تقييم مستويات microRNA222 والعامل النووي المرتبط بالعامل 2كمؤشرات حيوية محتملة لتشخيص وتحديد المرحلة السريرية لسرطان الغدة الدرقية الحليمي لدى المرضى العراقيين

Serum MicroRNA222 and Nuclear Factor Erythroid 2–Related Factor 2 As Potential Biomarkers for The Diagnosis and Clinical Staging of Papillary Thyroid Carcinoma in Iraqi Patients

Abstract

Papillary Thyroid Carcinoma (PTC) is the most common form of thyroid malignancy, yet accurate non-invasive diagnostic and prognostic biomarkers are still needed to complement conventional methods. MicroRNAs, particularly microR-222, are frequently dysregulated in various cancers, including thyroid cancer, where they regulate cell proliferation, differentiation, and apoptosis. Nuclear Factor Erythroid 2–Related Factor 2 (Nrf2) is a master regulator of antioxidant and detoxification responses, often aberrantly activated in cancer to promote survival and progression. This study aims to evaluate the diagnostic utility of serum miR-222 and Nrf2 levels in patients with PTC, and to investigate their potential correlation with the clinical staging and histopathological grading of the disease.
A case-control study was conducted from October 2024 to July 2025, involving a total of 87 participants from Safeer Al-Imam Al-Hussain (A.S) Surgical Hospital, Al-Kafeel Superspeciality Hospital and Al-Imam Al-Hussain Medical City in Kerbala City. The cohort comprised 16 patients with papillary thyroid carcinoma, 39 patients with benign thyroid diseases (including thyroid nodules and non-nodular diseases), and 32 apparently healthy control individuals. Patients with other types of thyroid cancer or non-specified thyroid diseases were excluded. Sociodemographic data (age, sex, smoking status, family history, job, disease duration, weight, height), clinical information (Ultrasound findings, Fine Needle Aspiration cytology reports), and pre-operative laboratory investigations (thyroid function tests, random blood sugar, HbA1c, complete blood count) were collected via a structured questionnaire. All patients underwent lobectomy or thyroidectomy, with final diagnoses confirmed by histopathological examination of formalin-fixed paraffin-embedded tissues. Ethical approval was obtained from relevant hospital and university ethics committees, and verbal consent was secured from all participants. For biomarker analysis, 5 mL of pre-operative venous blood was collected. Samples for microRNA-222 were placed in EDTA tubes and stored at -80°C. Samples for Nrf2 were collected in gel tubes, left for 15 minutes at room temperature, centrifuged at 4000 x g for 10 minutes to separate serum, and then aliquoted and stored at -80°C to prevent multiple freeze-thaw cycles. MicroRNA-222 signatures in all samples and control was analysis using TransScript Green One-step RT/Rl Enzyme Mix (Korea) was used for total RNA measurement according to the manufacturer’s instructions. Total RNA was purified using an RNA Clean XP Kit and RNase-Free DNase Set, and Nrf2 levels were measured by ELISA.
Results were shown that Serum Nrf2 and Folding Change microRNA222 levels were significantly lower in the combined patient group (benign + PTC) compared to apparently healthy controls. Conversely, Ct.microRNA222 values were higher in patients, confirming a lower abundance of microRNA222.
Nrf2 levels were consistently low in both benign and malignant groups compared to controls, showing no significant difference between benign and malignant conditions. However, Folding Change microRNA222 was drastically reduced in the malignant group compared to both healthy controls and benign cases, suggesting its potential to distinguish malignancy. When comparing patient outcomes, Nrf2 levels were similarly low in both prognosis and cure groups relative to healthy controls. Crucially, Folding Change microRNA222 was significantly lower in the ‘prognosis’ group compared to both healthy controls and the ‘cure’ group, where levels were comparable to healthy individuals. This indicates Folding Change microRNA222’s strong potential as a prognostic marker.
Nrf2 levels were significantly lower across all thyroid pathological subtypes (Hashimoto’s, multinodular goiter, nodular hyperplasia, solitary adenomatous nodule) compared to apparently healthy controls, implying a general impairment in antioxidant defense in thyroid diseases. In contrast, Folding Change microRNA222 was distinctly higher in Hashimoto’s Thyroiditis compared to all other groups, and lowest in solitary adenomatous nodule , highlighting its diverse roles across specific pathologies.
The Diagnostic Performance of the biomarkers (Nrf2, microRNA222-Ct, Folding Change microRNA222) showed limited discriminatory power (AUCs between 0.51-0.61) for differentiating cured patients from apparently healthy controls. Folding Change microRNA222 had the highest sensitivity (75%) but lowest specificity (50%).
Conclusion: This study demonstrates that serum Nrf2 levels are generally diminished across various thyroid pathologies, suggesting a widespread compromise in antioxidant defense. Serum microR-222, as indicated by “Folding Change microRNA222,” exhibits a more nuanced role, being significantly downregulated in PTC and poorer prognosis, and notably upregulated in Hashimoto’s Thyroiditis. While the overall diagnostic accuracy for distinguishing cured patients from apparently healthy controls was modest for the individual markers, the distinct patterns of microR-222 expression across different thyroid conditions, particularly its significant reduction in malignancy and poor prognosis, underscore its potential as a valuable, specific biomarker for diagnosis and prognostic assessment in Papillary Thyroid Carcinoma. Further research is warranted to validate these findings and explore their clinical integration.