The whole profiling and competing endogenous RNA network analyses of noncoding RNAs in adipose stem cells from diabetic, old and young patients
Authors: Ren Sen , xiong Hewei , Chen Jing , Yang Xiaofan , Liu Yutian , Guo Jiahe , Jiang Tao , Xu Zhao , Yuan Meng , Liu Yang , Zhou Nan , Chen Hongrui , Li Wenqing , Machens Hans-Günther , Chen Zhenbing 
Published: January 2021

Abstract
BackgroundMesenchymal stem cells including adipose stem cells (ASCs) have a huge potential in the field of translational medicine. Unfortunately, multiple factors including older age, co-existing diabetes and obesity may impair cellular function, which hinders the overall effectiveness of autologous stem cell therapy. Noncoding RNAs including microRNAs (miRNA), long ncRNAs (lncRNAs), and circular RNAs (circRNAs) have been demonstrated to play an important role in stem cell biology. However, the whole expression pattern and interaction of these RNAs in ASCs related to diabetes and aging remain unknown.MethodEdU, transwell and β-galactosidase staining assays were performed to assess the proliferation, migration and senescence of ASCs isolated from diabetic (D-ASCs), old (O-ASCs), and young (Y-ASCs) donators. The abilities of these ASCs modulating endothelial cells and fibroblasts functions were evaluated by tube formation and wound scratch assays. We conducted high-throughput RNA sequencing (RNA-seq) in these ASCs to uncover the differentially expressed (DE) RNAs. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein-protein interactions (PPI) analyses were performed to interpret the mRNAs with significant differences. The lncRNAs or circRNAs associated competing endogenous RNA (ceRNA) networks were constructed based on the bioinformatic analyses and real-time polymerase chain reaction (RT-PCR) results. The miR-145-5p mimics were transfected into old ASCs and verified by PCR.ResultsASCs from diabetic and old donators showed inferior migration ability and increased cellular senescence. Besides, old ASCs have decreased capacities for promoting endothelial cells angiogenesis and fibroblasts migration comparing to young ASCs. The DE miRNAs, mRNAs, lncRNAs and circRNAs were successfully identified by RNA-seq in O-ASCs vs Y-ASCs and D-ASCs vs O-ASCs. GO and KEGG analyses demonstrated DE mRNAs were significantly enriched in aging and cell senescence terms separately. PPI networks performed critical DE mRNAs in above groups. MiRNAs with high fold change and low p value were validated by PCR. The four ceRNA networks were conducted based on the bioinformatic analyses and validated miRNAs. The selected mRNAs, lncRNAs and circRNAs in PPI and ceRNA networks were further evaluated by PCR. Then, the ceRNA subnetworks were constructed based on above validated RNAs. In addition, the lncRNA RAET1E-AS1 - miR-145-5p - WNT11/BMPER axis was selected and validated by the PCR and correlation analyses. Finally, overexpression of miR-145-5p could rejuvenate old ASCs phenotype and augment their abilities for modulating endothelial cells and fibroblasts functions.ConclusionTaken together, our research may provide new clues to unveil the underlying mechanisms of ASCs dysfunction, and disclose novel targets for restoring their therapeutic properties.