Expression And Functional Analysis Of MicroRNAs In Bone Marrow Mesenchymal Stem Cells From Aged Rats

Adult stem cells are essential for maintaining tissue homeostasis and organ regeneration.The destruction of tissues and organs in aged individuals is closely associated with the reduction in quantity and function of adult stem cells.Mesenchymal stem cells?MSCs?are one of the most widely studied and clinically applicable adult stem cells.However,in the process of individual aging,the senile microenvironment in vivo plays an important role in stem cell senescence.The quantity of MSCs decreases with increasing age of the donor,accompanied by the decline in proliferation and viability.MSCs from elderly individuals at the early passage in the cultivation in vitro manifest senescent characteristics.This leads to declined organ function in elderly individuals and restrains the efficacy of autologous stem cell transplantation.Therefore,how to maintain or rejuvenate senescent MSCs is a key issue that deserves urgent exploration.Micro RNAs?miRNAs?derive from non-coding regions or introns of coding regions and are mainly transcribed by RNA polymerases II and III.miRNAs can influence gene expression through complementary pairing with target m RNAs,and further participate in the regulation of multiple complex biological processes.Generally,a single miRNA can target on multiple m RNAs,and multiple miRNAs can also modulate a single m RNA simultaneously.As one of the epigenetic mechanisms,miRNAs are crucial for maintaining cell homeostasis and regulating cellular senescence.In the aging process of individuals,the total miRNA levels of organisms or tissues show a downward trend,which may be closely related to the age-related biological alterations mediated by miRNAs.From C.elegans to humans,miRNAs play important regulatory roles in the aging process of multiple organisms.These miRNAs directly involved in aging regulation are called senescence-related miRNAs.Thus,we speculate that there might be some miRNAs which can exert modulatory effects on MSC senescence.However,it is still unclear which miRNAs present significantly differential expression in senescent MSCs,and what biological functions might be manipulated by these miRNAs.Objectives:To investigate the expression of miRNAs in MSCs from aged rats,and to perform functional enrichment analyses of target genes of differentially expressed miRNAs to determine their roles in stem cell senescence,so as to provide experimental evidences for exploring the molecular mechanisms of stem cell senescence and exploiting anti-aging interventions using miRNAs as therapeutic targets.Methods:In the present study,MSCs at passage 3?P3MSCs?were obtained from Wistar rats at 1-2 and 15-18 months of age by whole bone marrow adherent method and consecutive cultivation in vitro.Then MSC senescence was evaluated by cellular morphology,SA-?-gal staining,intracellular ROS content,and the expression of senescence-associated factors.Furthermore,miRNA sequencing?miRNA-seq?was performed based on young and senescent MSCs to determine the expression profiles of miRNAs during the process of MSC senescence.Finally,the target genes of differentially expressed miRNAs were predicted,and GO and KEGG Pathway enrichment analyses were performed.Results:1.Compared with young rats,P3MSCs from aged rats presented significant“senescence-like”alterations in morphology,such as irregular cell bodies and unclear boundary,along with increased cell area and decreased aspect ratio.Both the positive ratio of SA-?-gal staining and intracellular ROS content were elevated, and m RNA levels of the senescence-associated factors P16^INK4a and Rb1 were also obviously up-regulated.Thus,MSC natural senescence model was established.In subsequent experiments,P3MSCs obtained from young rats were used as young cells and P3MSCs from aged rats as senescent cells.2.miRNA-seq results showed that a total of 163 miRNAs were differentially expressed in senescent MSCs when compared to young MSCs,including 77 up-regulated and 86 down-regulated genes.Among them,the expression of miR-490-5p,miR-490-3p,miR-433-3p,miR-547-3p,miR-434-5p,miR-341-5p,miR-592-5p,miR-127-3p,miR-96-5p and miR-127-5p were remarkably elevated in senescent MSCs,while miR-298-3p,miR-296-5p,miR-298-5p,miR-204-5p,miR-296-3p,miR-203b-3p,miR-224-3p,miR-452-3p,miR-488-3p and miR-452-5p were significantly down-regulated.3.Based on the target gene prediction of the top ten differentially expressed miRNAs in senescent MSCs,we respectively obtained 591 target genes from up-regulated miRNAs and 1260 target genes from down-regulated miRNAs.And the number of common target genes of down-regulated miRNAs was strikingly higher than that of common target genes of up-regulated miRNAs.4.GO and KEGG Pathway enrichment analyses of target genes from key miRNAs displayed that the pathways such as MAPK signal pathway,Gn RH pathway,FOXO pathway,and c AMP pathway were mainly influenced by up-regulated miRNA,while the target genes of down-regulated miRNA were mostly enriched in the regulation of biological processes on cellular metabolism,nitrogen compound metabolism and cell development.Conclusions:1.MSCs from aged rats presented senescent alterations.Both the positive ratio of SA-?-gal staining and intracellular ROS content were elevated,and m RNA levels ofP16^INK4a and Rb1 were also up-regulated.2.A total of 163 miRNAs were differentially expressed in senescent MSCs,including 77 up-regulated and 86 down-regulated genes.3.The target genes of top ten differentially expressed miRNAs are mainly involved in the regulation of MSC senescence by affecting certain biological processes and pathways,such as cell metabolism,MAPK pathway and FOXO pathway.