miRNA Isoform Spectrum Expansion by Overexpression May Affect Target Gene Silencing

Abstract MicroRNAs (miRNAs) regulate gene expression primarily through translational inhibition and mRNA degradation. Their functional complexity is increased by the presence of various mature isoforms (isomiRs). These isomiRs add diversity to miRNA targeting and regulation, yet their impact on cellular systems remains insufficiently understood. In this study, we systematically assessed the effects of miRNA overexpression on the isomiR repertoire of five miRNAs (mir-34a, mir-129, mir-873, mir-133b, mir-7) across four different human cell lines (HeLa, HEK293T, LUHMES, and SH-SY5Y). Our results reveal a pronounced heterogeneity of isomiR profiles, which expanded significantly under overexpression conditions, with up to 845 unique isoforms detected per miRNA. Through time-course and dose-response experiments, we observed that the isomiR diversity increased rapidly within 8 hours following transfection and was sensitive to the amount of miRNA precursor transfected. Proteomic analysis indicated that changes in the isomiR repertoire might affect the regulatory capacity of miRNAs, altering the amplitude and frequency of target repression. Our findings underscore the complexity of miRNA regulation and highlight potential pitfalls in interpreting overexpression experiments, which may artificially expand the isomiR landscape and consequently modify the targetome. This study calls for careful consideration of isomiR dynamics in miRNA research and therapeutic development.