Identification and Characterization of a Long Noncoding RNA Involved in Osteogenic Differentiation
Anna Luan, MD, Ryan A. Flynn, M.D., Ph.D., Maxwell R. Mumback, B.S., Elizabeth R. Zielins, M.D., Elizabeth A. Brett, M.S., Charles P. Blackshear, M.D., Arash Momeni, M.D., Michael T. Longaker, M.D., M.B.A., Howard Y. Chang, M.D., Ph.D., Derrick C. Wan, M.D..
Stanford University, Stanford, CA, USA.
Purpose: Epigenetic regulation plays a key role in specification of cell fate and long non-coding RNAs (lncRNAs) have been shown to regulate chromatin patterns and large scale transcriptional profiles. Transcriptome analyses have specifically identified multiple candidate lncRNA which may guide bone differentiation. What role these may play and how they function to promote osteogenesis, however, remains poorly understood.
Methods: RNA libraries were generated from human ASCs and iPSCs undergoing osteogenic differentiation. Gene Ontology (GO) term analysis was performed for differentially-expressed transcripts and guilt-by-association analysis was used to identify lncRNA functionality. Transcriptome integration revealed one lncRNA with strong correlation to skeletal development. Osteogenic differentiation following siRNA-mediated suppression of this lncRNA was determined. Subcellular location of the lncRNA was also investigated by nuclear-cytoplasmic fractionation. Finally, Chromatin isolation by RNA purification (ChIRP) and mass spectrometry was used to determine functional protein binding partners to elucidate potential mechanism.
Results: Transcriptome integration and GO-term analysis identified lncRNA LOC100505806 with unidirectional upregulation during osteogenesis. Confirmation was obtained by qRT-PCR and knock-down in ASCs significantly reduced bone differentiation, as shown by histological staining and gene expression. Subcellular localization revealed LOC100505806 to be predominantly nuclear. Twenty interacting protein partners were identified using ChIRP and mass spectrometry. GO term analysis revealed the proteins to be involved in mRNA processing/stability, RNA localization/splicing, and regulation of gene expression.
Conclusions: lncRNAs may regulate large-scale transcriptional profiles and represent potential targets to manipulate osteogenic differentiation in multipotent stem cells. These findings also deepen our understanding of regulatory mechanisms guiding acquisition of cell fate.
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