Discovery and analytical assessment of urinary miRNA biomarkers for cervical cancer using advanced small RNA sequencing

Cervical cancer is primarily caused by persistent infection with high-risk human papillomavirus (hrHPV), which alters host RNA expression, including regulatory microRNAs (miRNAs). Because urine is emerging as a convenient biosource for cancer detection, we evaluated the feasibility of urinary miRNAs as non-invasive biomarkers for cervical cancer. We optimized miRNA isolation from whole urine and urine fractions using commercial RNA extraction kits, spike-in controls, and digital nanoplate PCR. Following identification of optimal conditions, IsoSeek small RNA sequencing was performed to profile miRNA expression in urine from women with cervical cancer (n = 20) and age-matched controls (n = 20). Differentially expressed candidates were then analytically confirmed by RT-qPCR. The Norgen RNA isolation kit applied to whole urine provided the most robust miRNA recovery. Sequencing identified significantly altered urinary levels of hsa-miR-143-3p and hsa-miR-204-3p in cervical cancer. The ratio of these miRNAs showed strong discriminatory performance for detecting cervical cancer using IsoSeek (AUC = 0.93) and remained robust after RT-qPCR analysis in the same cohort (AUC = 0.84). These findings support the feasibility of using urinary miRNA profiling for non-invasive biomarker discovery and highlight the hsa-miR-143-3p/hsa-miR-204-3p ratio as a promising candidate marker for cervical cancer detection.