MiR-34a-HK1 Axis Regulates Mesenchymal Stem Cells Senescence By Affecting Glucose Metabolism

Adult stem cells senescence is the major reason for individual aging,tissue organ aging and various age-related diseases.The destruction of tissues and organs in aging individuals is closely associated with the reduction in quantity and function of adult stem cells.As the superior seeding cells for tissue engineering and regenerative medicine,mesenchymal stem cells(MSCs)have decreased proliferation activity and cell viability,and attenuated multilineage differentiation potential with serial passages in vitro.This restricts the efficacy of autologous stem cell transplantation,and it also leads to the limitations of clinical application.Therefore,converting senescent stem cells into youth via regulation in order to improve age-related phenotypes and treat age-related diseases is an essential scientific issue in the fields of gerontology and stem cell research.Our previous studies have indicated that mi R-34 a expression was upregulated in senescent MSCs,while intracellular NAD content was reduced.And increased mi R-34 a promoted MSC senescence by reducing NAD level.As an essential coenzyme for cell energy transformation,NAD can participate in a variety of metabolic pathways,such as glucose metabolism.The conversion of NAD into NADH and NAD regeneration are implicated in glucose metabolic pathways.NAD depletion is closely related to cellular senescence and energy metabolic disorders.Many studies have reported that stem cell metabolism mainly depends on glycolysis.Mi R-34 a has potential binding sites for multiple glucose metabolism-related genes,such as hexokinase 1(HK1)and LDHA.We therefore postulate that mi R-34 a might affect glucose metabolism by targeting metabolism-related genes,and then modulates MSC senescence.In the present study,MSCs at passage 2(P2)and 10(P10)were obtained from male Wistar rats at 1-2 months of age by whole bone marrow adherent method and serially expanded culture in vitro,and MSC replicative senescence model was established.Then the major metabolic pathway and the levels of glucose metabolism in senescent MSCs were determined by glucose uptake,lactate secretion,ATP contentand ECAR levels.In order to screen out the differentially expressed genes related to glucose metabolism and find out the metabolic alterations in MSC senescence,gene expression profiles and the metabolites in young and senescent MSCs were explored by RNA sequence and metabolomics techniques.Furthermore,the effects of mi R-34 a on MSC senescence and cellular glucose metabolism were investigated by gene overexpression.Moreover,the bioinformatics prediction and dual-luciferase reporter gene assay were utilized to ascertain the direct targets of mi R-34 a.And the expression of HK1 affected by mi R-34 a was detected by RT-q PCR technique.Finally,the effects of HK1 on MSC senescence and cellular glucose metabolism,as well as mi R-34 a rescue assay were also explored to further clarify its potential regulatory mechanism.The results are as follows:1.Compared with P2 MSCs,P10MSCs presented significant changes in morphology,such as unclear boundary,increased cell areas and decreased aspect ratio.The positive ratio of SA-?-gal staining was increased and the m RNA expression of the senescence-associated factors such as P16INK4 a and Rb1 were also obviously elevated.We used P2 MSCs as young cells and P10 MSCs as senescent cells in the following experiments.2.The glucose uptake,lactate secretion,ATP content and ECAR level were significantly lower in senescent MSCs than those in young MSCs.After the treatment of 2-DG,the levels of glucose metabolism in both cells were markedly inhibited.3.RNA seq data showed that there were 868 up-regulated and 2006down-regulated genes in senescent MSCs when compared to young MSCs.GO and KEGG Pathway enrichment analyses indicated that biological processes and pathways such as tissue development and cell differentiation were mainly enriched in senescent MSCs,while cell metabolism,primal metabolism and cofactor metabolism were diminished in these cells.Combined with the above results,the glucose metabolism-related genes including HK1,PFK1 and PK were identified.4.Compared with young MSCs,22 significantly different metabolites in senescent MSCs were screened out by metabolomics techniques.Pathway analysis showed that lysine degradation and purine metabolism were significantly reduced in senescent MSCs,while the unsaturated fatty acid production pathway was obviously up-regulated.After metabolic pathway analysis using metabolites data combined withRNA seq differential genes,we found that fructose and mannose metabolism and glycolysis/gluconeogenesis manifested distinct alterations.5.Mi R-34 a in senescent MSCs was obviously elevated compared with young MSCs.Lentivirus-mediated mi R-34 a overexpression induced young MSCs senescence,with increased ratio of SA-?-gal positive cells and up-regulated expression of senescence-related factors.Moreover,the levels of glucose metabolism including glucose intake,lactate sretion,ATP contents and ECAR levels were evidently reduced in mi R-34 a over-expressed MSCs.6.Bioinformatics prediction demonstrated that there are potential complementary binding sites between mi R-34 a and the 3?-UTR region of HK1 m RNA.Dual luciferase reporter gene assay further verified that HK1 was a direct target of mi R-34 a.In addition,HK1 m RNA expression was down-regulated by mi R-34 a overexpression.7.Overexpression of HK1 up-regulated the level of glucose metabolism in senescent MSCs.The cell body became slender and smaller,and the positive ratio of SA-?-gal staining was decreased.8.Co-expression of mi R-34 a and HK1 in young MSCs can up-regulate the lower HK1 m RNA level induced by mi R-34 a overexpression,and inhibit mi R-34 a mediated MSC senescence.In summary,MSCs depend mostly on glycolysis for ATP production.The levels of glucose metabolism in senescent MSCs are significantly decreased.HK1 was a direct target of mi R-34 a,and mi R-34a-HK1 axis could regulate MSC senescence by affecting glucose metabolism.The present study reveals the regulatory mechanism of mi R-34 a on stem cell senescence from the perspective of cell metabolism and epigenetics.It will provide an experimental foundation for obtaining sufficient quantity of seeding cells,and create a novel field for delaying stem cell senescence.