microRNA - 125 b Promotes Neuronal Differentiation in Human Cells by 1 Repressing Multiple Targets

Running title: miR-125b promotes neuronal differentiation 21 ABSTRACT 1 microRNAs are a class of small non-coding RNAs that regulate gene expression at the 2 post-transcriptional level. Research on microRNAs has highlighted their importance in neural 3 development but the specific functions of neural-enriched microRNAs remain poorly understood. 4 We report here the expression profile of microRNAs during neuronal differentiation in the human 5 neuroblastoma cell line SH-SY5Y. Six microRNAs were significantly upregulated during 6 differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We 7 demonstrated that ectopic expression of either miR-124a or miR-125b increases the percentage of 8 differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional 9 analysis on miR-125b and demonstrated the important role of this miRNA in both spontaneous and 10 induced differentiation of SH-SH5Y cells. miR-125b is also upregulated during differentiation of 11 human neural progenitor ReNcell VM cells, and miR-125b ectopic expression significantly 12 promotes neurite outgrowth of these cells. To identify the targets of miR-125b regulation, we 13 profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y 14 cells. miR-125b represses 164 genes that contain the seed match sequence of the microRNA and/or 15 predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that 16 a subset of miR-125b-repressed targets antagonize neuronal genes in several neurogenic pathways, 17 thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further 18 validated the binding of miR-125b to the microRNA response elements of ten selected mRNA 19 targets. Together, we report here for the first time the important role of miR-125b in human neuronal 20 differentiation.