Gene Expression Profile Changes After Short-activating RNA-mediated Induction of Endogenous Pluripotency Factors in Human Mesenchymal Stem Cells

Abstract

It is now recognized that small noncoding RNA sequences have the ability to mediate transcriptional activation of specific target genes in human cells. Using bioinformatics analysis and functional screening, we screened short-activating RNA (saRNA) oligonucleotides designed to target the promoter regions of the pluripotency reprogramming factors, Kruppel-like factor 4 (KLF4) and c-MYC. We identified KLF4 and c-MYC promoter-targeted saRNA sequences that consistently induced increases in their respective levels of nascent mRNA and protein expression in a time- and dose-dependent manner, as compared with scrambled sequence control oligonucleotides. The functional consequences of saRNA-induced activation of each targeted reprogramming factor were then characterized by comprehensively profiling changes in gene expression by microarray analysis, which revealed significant increases in mRNA levels of their respective downstream pathway genes. Notably, the microarray profile after saRNA-mediated induction of endogenous KLF4 and c-MYC showed similar gene expression patterns for stem cell- and cell cycle-related genes as compared with lentiviral vector-mediated overexpression of exogenous KLF4 and c-MYC transgenes, while divergent gene expression patterns common to viral vector-mediated transgene delivery were also noted. The use of promoter-targeted saRNAs for the activation of pluripotency reprogramming factors could have broad implications for stem cell research.

Identification of Small Activating RNAs that Enhance Endogenous OCT4 Expression in Human Mesenchymal Stem Cells

“…Our results indicate that RNAa-mediated OCT4 activation works consistently in a panel of human MSCs that we tested. Other groups have also shown that promoter-targeted saRNAs could consistently induce endogenous KLF4 and c-MYC expressions in human MSCs derived from bone marrow [33]. Taken together, these observations suggest that saRNA-mediated gene activation could effectively upregulate endogenous pluripotent factors in human MSCs.…”

The role of antisense long noncoding RNA in small RNA-triggered gene activation

“…Similarly, small RNA-induced TGA has been reported in mammalian systems Watts et al 2010;Voutila et al 2012;Matsui et al 2013;Reebye et al 2013a,b), though the molecular mechanisms have not been fully elucidated. In these previous studies, TGA has been induced by two or more shRNAs that target the promoter sequence (Chu et al 2012).…”

“…Despite these and other demonstrations of mammalian TGA (Place et al 2008;Matilainen et al 2010;Matsui et al 2010;Voutila et al 2012;Reebye et al 2013a,b), the molecular mechanisms of the process are still not well understood, including the details about the role of lncRNAs in TGA and whether they act in an allele-specific cis-mode or a trans-mode. In this study, we devised a TGA model based on CMV-EGFP reporter gene system to investigate the factors required for TGA.…”

RNA Activation

“…[ 7 ]. This concept, again, is subjective, as there is evidence to show that transcription factors that are able to drive pluripotency can be induced by saRNAs in human mesenchymal stem cells [ 37 ]. The Ago2 processing unit has also created some dispute, where it is thought that Ago2-induced degradation of the strand does not emerge on the noncoding transcript of the target gene.…”

“…The ability to modulate pluripotent reprogramming factors, such as Kruppel-like factor 4 (KFL4) and c-Myc, by synthetic saRNA is likely to accelerate stem cell research [ 37 ]. Combined expression of OCT3/4, SOX2, KLF4, and c-Myc already allows reprogramming of adult fi broblasts into induced pluripotent stem cells for a myriad of applications [ 42 ].…”