Exploratory Small RNA Sequencing in Localized Versus Locally Advanced Prostate Cancer: Putative Roles of CLTC and CDK6 as Downstream Targets of Differentially Expressed MicroRNAs.

Background/Objectives: Prostate cancer (PCa) remains a leading cause of cancer-related mortality in men. While localized PCa carries a favorable prognosis, progression to locally advanced disease substantially worsens outcomes. There is an unmet need for molecular markers that reliably distinguish indolent from aggressive tumors. MicroRNAs (miRNAs), small non-coding RNAs that post-transcriptionally regulate gene expression, are implicated in cancer development and progression, and their expression profiles may serve as putative biomarkers of disease stage. Methods: We performed small RNA sequencing on formalin-fixed paraffin-embedded (FFPE) prostate tumor tissues from nine patients (five localized, four locally advanced-stage). Differentially expressed miRNAs were identified using a bioinformatics pipeline (fold-change ≥ 2, p < 0.05). Target gene prediction, KEGG pathway enrichment, and miRNA-mRNA interaction network analyses were conducted, followed by in silico validation using two independent public mRNA datasets (GSE46602 and GSE21034, a subseries of GSE21032). Results: Seven unique mature miRNAs were differentially expressed between localized and locally advanced PCa. Five miRNAs (miR-145-5p, miR-205-5p, miR-206, miR-24-1-5p, and hsa-miR-3648) were downregulated in locally advanced tumors, while hsa-miR-625-3p and miR-324-5p were upregulated. Network analysis identified CLTC and CDK6 as putative downstream targets co-targeted by multiple downregulated miRNAs. Independent validation using GSE46602 and GSE21034 consistently demonstrated significantly higher expression of CLTC and CDK6 in locally advanced prostate cancer following Benjamini-Hochberg correction. Conclusions: This exploratory pilot analysis identified several miRNAs that differ between localized (pT2) and locally advanced (pT3-T4/N+) prostate cancer. Independent validation across two publicly available mRNA cohorts supports CLTC and CDK6 as candidate stage-associated genes. However, these findings remain hypothesis-generating and require validation in larger prospective cohorts together with functional confirmation before biomarker or therapeutic claims can be made.