Clinical Evaluation of miR-135b-5p and miR-21-5p Expression on Self-Collected Cervical Swab Samples for Triaging HR-HPV-Positive High-Grade Preneoplastic Lesions in India

Objective: This study aimed to evaluate the triage performance of microRNA (miRNA) biomarkers on self-collected, high-risk human papillomavirus (HR-HPV)-positive cervical swabs to discern cervical intraepithelial neoplasia II–III or high-grade squamous intraepithelial lesions (HSIL) in Indian women for precise diagnosis and treatment prioritization. Methods: Through in silico analysis, miR-135b-5p and miR-21-5p were identified as upregulated and amplified miRNAs in cervical cancer (CaCx). Quantitative real-time PCR was used to assess miR-135b-5p and miR-21-5p and their targeting, LIM domain-containing protein 1 (LIMD1) and von Hippel–Lindau (VHL) messenger RNA (mRNA) expression, in a discovery cohort ( N = 42; normal cervical epithelium [ N = 9], CIN [ N = 12], and CaCx [ N = 21] tissue samples). Thereafter, miRNA expression was validated in an independent set of self-collected cervical swabs ( N = 243; HR-HPV-positive [asymptomatic ( N = 52), low-grade squamous intraepithelial lesions (LSIL) ( N = 50), and HSIL ( N = 52), CaCx ( N = 35)] and HPV-negative normal [ N = 54]). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic performance of these two candidate miRNAs. Results: In discovery-phase samples, miR-135b-5p showed progressive upregulation from CIN ( p = 0.056) to CaCx ( p = 0.012), while miR-21-5p showed significant overexpression at CIN ( p = 0.011) and CaCx ( p = 0.005) compared to normal tissue. Concurrently, significant downregulation of LIMD1/VHL mRNA expression was observed in the same CaCx samples. In the validation cohort, miR-135b-5p and miR-21-5p were significantly overexpressed in HSIL samples compared to lower-grade lesions (≤LSIL), with a corresponding downexpression of LIMD1/VHL. ROC curve analysis demonstrated high sensitivity and specificity for miR-21-5p and miR-135b-5p—both individually and in combination—for distinguishing HR-HPV + HSIL from LSIL, asymptomatic, or HPV-negative normal samples. Conclusion: miR-135b-5p and miR-21-5p constitute a feasible triage biomarker on noninvasive, self-collected cervical swab samples for early, precise diagnosis of women with HR-HPV-positive HSIL, requiring treatment prioritization in low-resource settings.