MicroRNAs (miRNAs) are small non-coding RNAs that play pivotal roles in post-transcriptional gene regulation, influencing development, differentiation, and disease pathogenesis. Since their discovery in 1993, miRNAs have been recognized for their evolutionary conservation and pleiotropic effects, with the 2024 Nobel Prize underscoring their significance in post-transcriptional regulation via the RNA interference (RNAi) pathway. This review synthesizes the complete life cycle of miRNAs—from transcription and processing to function and decay—emphasizing regulatory mechanisms and their implications in human diseases, particularly cancer. We discuss how epitranscriptomic modifications influence miRNA biogenesis and activity, explore their nuclear and mitochondrial functions, and address emerging challenges in miRNA-based therapeutics, including the expanding small RNA landscape such as tRNA-derived small RNAs (tsRNAs), and Argonaute (AGO)-independent activities. Despite hurdles such as modest multi-target effects, off-target interactions, and delivery challenges, miRNAs remain promising as both biomarkers and therapeutic agents, underscoring the need for sustained research to bridge preclinical insights with clinical applications.

MicroRNAs (miRNAs) are small non-coding RNAs that play pivotal roles in post-transcriptional gene regulation, influencing development, differentiation, and disease pathogenesis. Since their discovery in 1993, miRNAs have been recognized for their evolutionary conservation and pleiotropic effects, with recent Nobel Prize awards underscoring their significance in RNA interference (RNAi). This review synthesizes the complete life cycle of miRNAs —from transcription and processing to function and de-cay— emphasizing the regulatory mechanisms that govern these processes and their implications in human diseases, particularly cancer. We discuss how epitranscriptomic modifications influence miRNA biogenesis and activity, explore their nuclear and mi-tochondrial functions, and address emerging challenges in miRNA-based therapeutics, including the expanding small RNA landscape, such as tRNA-derived small RNAs (tsRNAs), and Argonaute (AGO)-independent activities. Despite hurdles such as modest multi-target effects and unvalidated specificity, miRNAs remain promising as both biomarkers and therapeutic agents, underscoring the need for sustained research to bridge preclinical insights with clinical applications.