Knockdown Of Long Noncoding RNA NONHSAT035482.2 Rejuvenates Aged Adipose-derived Stem Cells Via MiR-143-3p/ADD3 Axis

Background: Mesenchymal 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 micro RNAs(mi RNA),long nc RNAs(lnc RNAs),and circular RNAs(circ RNAs)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 aging remain unknown.Method: Ed U,transwell and ?-galactosidase staining assays were performed to assess the proliferation,migration and senescence of ASCs isolated from 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),KEGG pathway,and protein-protein interactions(PPI)analyses were performed to interpret the m RNAs with significant differences.The lnc RNAs or circ RNAs associated competing endogenous RNA(ce RNA)networks were constructed based on the bioinformatic analyses and quantitative polymerase chain reaction results.Lnc RNA NONHSAT035482.2 was screened out from differentially expressed lnc RNAs validated by real-time quantitative RT-PCR.Dual-luciferase assays,lentivirus transduction and database predication were used to identify the mechanism and function of RNAs.Results: ASCs from 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 mi RNAs,m RNAs,lnc RNAs and circ RNAs were successfully identified by RNA-seq in O-ASCs vs Y-ASCs.GO and KEGG analyses demonstrated DE m RNAs were significantly enriched in aging and cell senescence terms separately.PPI networks performed critical DE m RNAs in above groups.mi RNAs with high fold change and low p value were validated by PCR.The selected m RNAs,lnc RNAs and circ RNAs in PPI and ce RNA networks were further evaluated by PCR.Then,the ce RNA subnetworks were constructed based on above validated RNAs.Finally,lnc RNA NONHSAT035482.2 was upregulated in aged ASCs and regulated cellular senescence of ASCs.Knockdown of lnc RNA SAN and silencing ADD3 in ASCs showed increased ASC function,inhibition of cellular senescence and promoting effects of CM on endothelial cells as well as fibroblasts.Mechanistically,the SAN can sponge mi R-143-3p to regulate the expression of ADD3,and silencing ADD3 could also rejuvenate the senescent ASCs.Application of SAN-depleted aged-ASCs accelerated wound closure in skin injury model compared with transplantation of aged-ASCs.Conclusion: Taken together,our research may provide new clues to unveil the underlying mechanisms of ASCs senescence and dysfunction,Lnc RNA NONHSAT035482.2 mediates ASC senescence by regulating mi R-143-3p/ADD3 pathway,indicating a potential target to rejuvenate senescent ASCs and enhance wound repair.