| Background and purpose:Tissue engineering is an important part in regenerative medicine. It generally includes seed cells, cellular scaffold and growth factors. Among the many kinds of seed cells,mesenchymal stem cells (MSCs) are the most widely used and have great developmental potential. MSCs are one kind of adult stem cells, and they have the ability of self-renewing and differentiating into multiple cell types. However, the clinical uses of MSCs are very rare. One of the reasons is that MSCs tend to become cellular senescent. Cellular senescence is a process in which cells stop cell division permanently while remain viable.Up till now, the mechanisms underlying the initiation and progression of cellular senescence are still not clearly understood. There have been reports showing that microRNAs may play important roles in cellular senescence. miRNAsare a group of small and non-coding RNAs that are 18-25 nucleotides in length and generally repress target gene expression by degrading mRNA or preventing translation. The results of our preliminary experiments suggested that the expression of miR-29c-3p was gradually increased during the replicative senescence of MSCs,indicating important roles of miR-29c-3p in MSC senescence. Our research aims to discuss the effects of miR-29c-3p on cellular senescence of MSCs by gene gain-and-loss function analysis, predict and verify the target gene of miR-29c-3p and explore the functional signal pathways. We hope to understand the mechanisms of cellular senescence of MSCs and pave the way for future application of MSCs.Methods: Human bone marrow-derived MSCs were subcultured for a long time in vitro. Besides, MSCs were transfected with miRNA mimic and inhibitors to increase and decrease the miR-29c-3p expression. Then, the senescence associated ?-galactosidase staining (SA-?-gal), cell immunofluorescence staining, Edu proliferation assay, CCK-8 proliferation assay, ELISA assay, qPCR, Western blot assay and cell differentiation capacity assay were done. The target gene of miR-29c-3p was predicted and verified through bioinformatics software and molecular biology methods. The small RNA interfering methods were used to inhibit the expression of target gene of miR-29c-3p. Then,senescence associated cellular phenotypes were tested.Results:1.After long-time subculture in vitro, MSCs showed positive SA-?-gal staining,senescence associated heterochromatin foci (SAHF),senescence associated secretory phenotype (SASP), expression changes of senescence marker genes and decreased differentiation ability.2. When miR-29c-3p was over-expressed in MSCs, MSCs showed positive SA-?-gal staining,SAHF,SASP,expression changes of senescence marker genes and impaired differentiation ability.3. When the miR-29c-3p expression was down-regulated in MSCs, MSCs showed decreased positive rate of SA-?-gal staining, decreased SAHF, contrary SASP, expression changes of senescence marker genes and relieved differentiation ability.4. The functional downstream target gene of miR-29c-3p was CNOT6.5.When the expression of CNOT6 was inhibited in MSCs, MSCs showed decreased positive rate of SA-?-gal staining, decreased SAHF, contrary SASP, expression changes of senescence marker genes and relieved differentiation ability.6. The p53-p21 and p16-pRB signal pathways were activated in replicative senescence and miR-29c-3p induced senescence of MSCs.Conclusions:1. MSCs entered into replicative senescence after long-time subculture in vitro.Senescent MSCs showed positive SA-?-gal staining, SAHF, SASP, expression changes of senescence marker genes and impaired differentiation ability.2.miR-29c-3p promoted the cellular senescence of MSCs.3.CNOT6 was the target gene of miR-29c-3p and could inhibit cellular senescence of MSCs.4.miR-29c-3p promoted cellular senescence of MSCs by activating p53-p21 and p16-pRB signal pathways... |
PDF Full Text Request