Human Antigen R Binding and Regulation of SOX2 mRNA in Human Mesenchymal Stem Cells

Abstract

Since 2005, sex determining region y-box 2 (SOX2) has drawn the attention of the scientific community for being one of the key transcription factors responsible for pluripotency induction in somatic stem cells. Our research investigated the turnover regulation of SOX2 mRNA in human adipose-derived stem cells, considered one of the most valuable sources of somatic stem cells in regenerative medicine. Mitoxantrone is a drug that acts on nucleic acids primarily used to treat certain types of cancer and was recently shown to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. In addition, mitoxantrone has been shown to inhibit the binding of human antigen R (HuR) RNA-binding protein to tumor necrosis factor-a mRNA. Our results show that HuR binds to the 39-untranslated region of SOX2 mRNA together with the RNAinduced silencing complex miR145. The HuR binding works by stabilizing the interaction between the 39-untranslated region and the RNA-induced silencing complex. Cell exposure to mitoxantrone leads to HuR detachment and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows improvement of the spheroid-forming capability of the adipose-derived stem cells. The silencing of HuR confirmed the above observations and illustrates how the RNA-binding protein HuR may be a required molecule for regulation of SOX2 mRNA decay.

HuR interacts with lincBRN1a and lincBRN1b during neuronal stem cells differentiation

“…RIP was performed as reported in Latorre et al 2016 [27] . Briefly, cells were cultured in the different experimental conditions (NSCs grown in floating conditions treated or not with DT, and differentiated NSCs treated or not with DT) and at the end of the treatments were syringed with a U-100 insulin needle in 500 µl lysis NT2 buffer (50 mM tris-HCl pH7.7, 150 mM NaCl, 1 mM MgCl2; 0.05% NP40, 1 U/µl RNase IN, 20 mM DTT, 1% BSA, Protease inhibitor cocktail) chilled at 4°C.…”

“…The RNA quality was assessed using a spectrophotometer (NANOPhotometer® NP80, IMPLEN) and visualized on agarose gel. Total RNA (1 μg) was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions and used as template for Real-Time RT-PCR analysis [27]. Real-Time RT-PCR was performed with StepOnePlus TM Real-Time PCR System (Thermo Fisher) using iQ SYBR Green Supermix (Bio-Rad).…”

“…They were then cloned into a pMirTarget plasmid (Origene, Rockville, MD). Plasmid 3ʹUTR SOX2 was used as control [27]. The Pierce Firefly Luciferase Flash Assay Kit (Sigma-Aldrich) was used according to manufacturer's instructions 24 h after transfection with Lipofectamine™ 2000 (Invitrogen).…”

“…HuR has also been reported to influence lincp21's decay in mouse fibroblasts, where this interaction has been proposed to regulate the translation of a subset of target mRNAs [25,26]. Furthermore, HuR is able to influence the stemness state of mesenchymal stem cells by regulating the mRNA turnover of the SOX2 master gene [27]. Less is known about the regulatory pathways in which HuR and lncRNAs are involved, and their molecular mechanisms of action, particularly in neural precursor cells' differentiation.…”

RNA-Targeted Therapies and High-Throughput Screening Methods

“…HuR binds to ARE-RNA of sex-determining region y-box2 (SOX2) and induces RNA degradation. Mitoxantrone disrupted the interaction between HuR and SOX2 mRNA, resulting in an upregulated level of SOX2 mRNA and protein in mesenchymal stem cells [137]. The same AlphaScreen assay was applied to a small-scale screen from 107 commercially available anti-inflammatory compounds [138].…”