Screening Of Age-related Circular RNAs In Rat Bone Marrow Mesenchymal Stem Cells And Regulatory Network Analysis

Aging is a chronic and complex process,closely associated with the permanent and progressive decline in cellular physiological functions.With the occurrence of aging,adult stem cells in the tissues decrease and their functions gradually weaken,which is one of the main reasons for the reduction in the capabilities of body's regeneration and repair.Bone marrow mesenchymal stem cells(MSCs)originate from the mesoderm,and consecutive cultures in vitro lead to cellular senescence.Cell proliferation ability decreases and cell cycle is blocked,as well as cellular differentiation potentials also attenuate.MSCs derived from aged rats also have similar characteristics.Therefore,it is essential for the clinical applications of stem cells to investigate the underlying molecular mechanisms that lead to MSC senescence and find strategies to delay aging to preserve stem cell functions.As a new type of endogenous non-codingRNAs,circularRNAs(circRNAs)can be used as a regulator of microRNA(miRNA)sponge,splicing and transcription,which play vital roles in modulating cellular senescence,cell apoptosis,the occurrence and development of tumors and age-related diseases.Currently,circRNAs have become one of research hotspots in the field of aging.It has been reported in the literature that with the occurrence of aging,circRNAs have essential regulatory effects on the aging of brain,muscle,skin and reproductive system.Thus,we speculate that some circRNAs might have modulatory effects on MSC senescence.However,it is still unknown which circRNAs present remarkable differential expressions in senescent MSCs,and which signaling pathways might be manipulated by these circRNAs.Objectives:To investigate the differential expressions of circRNAs in senescent MSCs,and select age-related circRNAs,and further analyze their functions and regulatory network,so as to explore the molecular biological mechanisms of MSC senescence and to provide experimental basis for applying circRNAs as targets to delay aging and finding the strategies to maintain stem cell functions.Methods:In this study,MSCs derived from 1-2 months and 15-18 months(young and old)rats at passage 3 were firstly obtained by the whole bone marrow adherent method and consecutive cultivation in vitro.Then,the morphological characteristics,surface markers,intracellular ROS level,osteogenic differentiation,and SA-?-gal activity and the expression of senescence-associated factors were examined.Furthermore,circRNA sequencing was performed to determine the expression profiles of circRNAs in young and senescent MSCs,and then age-related circRNAs were screened out.Lastly,circRNA-miRNA-mRNA co-expression network was set up for the significantly differentially expressed circRNAs,and Go and KEGG enrichment analyses of their target genes and protein interaction analysis were performed.Results:1.MSCs derived from young rats grew well with clear boundaries,long and fusiform in shape,while MSCs from old rats grew slowly,the cell body wasirregular,and the cytoplasm was granular.The cell surface area increased while the ratio of cellular length and width decreased.CD44 and CD105 were highlyexpressed in both groups,and CD31 and CD45 were negative.Compared with the young group,the osteogenic differentiation in the old group was obviously reduced,and intracellular ROS level,the positive rate of SA-?-gal staining and p16^INK4a and Rb1 expression were significantly elevated.Thereby,age-related senescent MSCs have been obtained,which will be used in the following experiments.2.The data analysis of circRNA sequencing indicated that there were 4,229 differentially expressed circRNAs between young and old MSCs.Approximately 95%of circRNAs were predicted to be less than 2,000 nt.Among the 4,229 circRNAs,4 circRNAs were markedly up-regulated and 25 circRNAs were obviously down-regulated.A total of 4,200 circRNAs were not significantly different.3.RT-q PCR was performed to verify 4 selected circRNAs with significant difference.The results demonstrated that the expression of circ-Phldb2 was extraordinarily up-regulated in age-related senescent MSCs,while the expressions of circ-Mdm2 and circ-Rab31 were down-regulated strikingly,which was consistent with the sequencing results.However,circ-Cd2ap expression was contradictory.4.The results of the circRNA-miRNA-mRNA co-expression network showed that circRNAs played a central role in this network,and a single circRNA could be associated with a variety of miRNAs,so as to further influence the functions ofmRNAs.5.The mRNAs from the circRNA-miRNA-mRNA interaction network were used in GO and KEGG Pathway analyses.The results exhibited that significant alterations were present in biological processes such as cellular metabolism,cellular macromolecular metabolism and organic substance metabolic process, and signaling pathways such as mitophagy,cancer pathways,lysosomes and autophagy.6.The interaction network of differentially expressed circRNA target proteins in age-related senescent MSCs manifested that there were eleven proteins closely related to other proteins,including Nup37,Hsp90aa1,Pafah1b1,Dync1li2,Sptan1,Prkca,Ntrk2,Ap2a2,Dnm2,Hip1 and Tgoln2.Conclusions:1.As rats age,MSCs undergo age-associated senescence.2.Using circRNA sequencing,three age-related circRNAs are screened out in senescent MSCs,namely circ-Phldb2,circ-Mdm2 and circ-Rab31.3.Cellular metabolism,cellular macromolecular metabolism and mitochondrial autophagy are significantly changed in age-associated senescent MSCs,which may play the important roles in the regulatory network of circRNAs on MSC senescence.