miR-449a is a critical factor regulating chondrogenesis of MSC via directly targeting LEF-1

Abstract

Relatively newly discovered molecule identified as microRNA has proven its critical role in various developmental processes over the years. Consisted of 20-24 nucleotide, the non-coding RNA molecule microRNAs recognize and inhibit expression of its target genes by either degrading its target mRNA transcript and/or by inducing inhibition of mRNA translation. Despite the increasing interest in identification of microRNAs involved in various processes, however, the study remains at premature stage in stem cell research and especially in human mesenchymal stem cell differentiation. As such, this study was aimed a) to identify a novel gene involved in chondrogenesis and b) to identify potential microRNAs regulating the expression of such identified gene. By comparing multiple culture condition and methods, we have identified micromass culture method showed the most effective chondrogenesis capacity. High throughput screening via microarray analysis was performed to detect both gene and microRNA expression distinctively present in MSCs cultivated in chondrogenic condition only. By thorough statistical analysis and microRNA target prediction, we have detected that LEF-1, lymphoid enhancer-binding factor 1, was uniquely expressed in MSCs cultivated in chondrogenic media and that multiple microRNAs predicted to target this gene showed positive expression profile as well. LEF-1 is an extensively studied gene known for its critical role during canonical pathway of Wnt signaling, and its positive effect on early stages of chondrogenesis has been well documented. Consensus agreements between microarray data, RT-PCR and quantitative realtime PCR have all indicated that the screened microRNAs in this study are highly likely to target LEF-1. Further study has revealed that one of these predicted microRNAs to target LEF-1, namely miR-449a directly binds to the seed sequence on 3’ UTR of LEF leading to direct repression of its expression. Furthermore, we have also identified, in perfect alignment with previous studies, that when LEF-1 expression was inhibited, the overall key chondrogenic marker genes such as Type II Collagen and SOX9, as well marker genes for maturation such as MMP13 and Type X collagen showed noticeable decrease. Collectively, the currently study successfully identified that miR-449a regulates chondrogenesis of BMSC via directly targeting LEF-1.