ABSTRACT
Long noncoding RNA (lncRNA) are regulatory RNAs >200 nt. We previously showed that lncRNA GAS5 decreases significantly in serum of type 2 diabetes mellitus (T2DM) patients. Hence, we sought to decipher the molecular mechanisms underlying the role of GAS5 in T2DM in adipose tissue. Using CHIP-RIP, we demonstrate that GAS5 binds to promoter of insulin receptor to regulate its expression, and its depletion inhibits glucose uptake and insulin signaling. Toward stabilizing GAS5 levels in T2DM, we incorporated a strategy to limit the degradation of GAS5 by blocking the interaction of GAS5 and UPF1 with a small molecule identified using OBTC screening strategy. NP-C86 binds to GAS5 with high affinity, and increases GAS5 levels and glucose uptake in diabetic patient adipocytes. As a broader impact, NP-C86 may be used as a molecular probe to investigate the intricacies of GAS5 in relevant biological systems as it offers specificity, efficient cellular uptake and is non-cytotoxic.
Subject(s)
Adipocytes/metabolism , Diabetes Mellitus, Type 2/pathology , RNA, Long Noncoding/metabolism , Small Molecule Libraries/metabolism , Adipose Tissue/cytology , Cell Differentiation , Diabetes Mellitus, Type 2/genetics , Gene Expression Regulation/drug effects , Glucose/metabolism , Humans , Insulin/metabolism , Promoter Regions, Genetic , RNA Interference , RNA, Long Noncoding/antagonists & inhibitors , RNA, Long Noncoding/genetics , RNA, Small Interfering/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Signal Transduction , Small Molecule Libraries/chemistry , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
BACKGROUND: Neuroinflammation is a common therapeutic target for traumatic brain injury (TBI) due to its contribution to delayed secondary cell death and has the potential to occur for years after the initial insult. Exosomes from adipose-derived stem cells (hASCs) containing the long noncoding RNA MALAT1 are a novel, cell-free regenerative approach to long-term recovery after traumatic brain injury (TBI) that have the potential to modulate inflammation at the genomic level. The long noncoding RNA MALAT1 has been shown to be an important component of the secretome of hASCs. METHODS: We isolated exosomes from hASC containing or depleted of MALAT1. The hASC-derived exosomes were then administered intravenously to rats following a mild controlled cortical impact (CCI). We followed the rats with behavior, in vivo imaging, histology, and RNA sequencing (RNA Seq). RESULTS: Using in vivo imaging, we show that exosomes migrate into the spleen within 1 h following administration and enter the brain several hours later following TBI. Significant recovery of function on motor behavior as well as a reduction in cortical brain injury was observed after TBI in rats treated with exosomes. Treatment with either exosomes depleted of MALAT1 or conditioned media depleted of exosomes showed limited regenerative effects, demonstrating the importance of MALAT1 in exosome-mediated recovery. Analysis of the brain and spleen transcriptome using RNA Seq showed MALAT1-dependent modulation of inflammation-related pathways, cell cycle, cell death, and regenerative molecular pathways. Importantly, our data demonstrates that MALAT1 regulates expression of other noncoding RNAs including snoRNAs. CONCLUSION: We demonstrate that MALAT1 in hASC-derived exosomes modulates multiple therapeutic targets, including inflammation, and has tremendous therapeutic potential for treatment of TBI.
