MiR-141-3p/PFKM Axis Regulates Mesenchymal Stem Cell Senescence By Affecting Glucose Metabolism

Mesenchymal stem cells(MSCs)are the basis of cell therapy for many diseases,which are a kind of adult stem cells that have been studied early and deeply.At present,MSCs have become the main seed cells of stem cell tissue engineering.Like somatic cells,stem cells will undergo morphological changes,proliferation and differentiation ability will also be reduced to varying degrees in the process of life,so stem cell senescence will lead to the aging of the body,resulting in the occurrence and development of various age-related diseases.Cellular senescence limits the clinical applications,such as autologous stem cell transplantation,tissue and organ regeneration and repair,etc.Therefore,anti-aging research of stem cells has become a hot direction in the field of geriatrics and stem cell research.Clarifying the regulatory mechanisms of stem cell senescence will contribute to maintaining the number and function of stem cells,which is of great significance for delaying aging and preventing and treating age-related diseases.Cellular senescence is accompanied by alterations in metabolic function.Carbohydrates are the main source of cellular energy metabolism.Glucose metabolism can be divided into glycolysis,mitochondrial oxidative phosphorylation,gluconeogenesis and pentose phosphate pathways.A large number of literatures show that the main metabolic mode of stem cells is glycolysis.Stem cells maintain the self-renewal and proliferation through glycolysis in a resting state to protect them from damage caused by oxidative stress.As a kind of non-coding RNA,mi RNAs can participate in the regulation of cellular senescence and metabolism.Combined with the preliminary experiments of our group,glycolysis is the main way of metabolism and energy supply of MSCs,and the glucose metabolism level of senescent MSCs is reduced.We propose that during the process of MSC senescence,certain genes related to glucose metabolism will change.These genes regulate the glucose metabolism level of MSCs and further affect cellular senescence.However,which genes related to glucose metabolism will change during the process of MSC senescence?What is the effect regulating the expression of differential genes on the glucose metabolism of MSCs?Can it further affect the senescence of MSCs?Are there specific mi RNAs that regulate genes related to glucose metabolism and influence cell metabolism and senescence?What is its possible mechanism?These questions remain unclear.Purpose:To screen and identify differentially expressed genes related to glucose metabolism in senescent MSCs,clarify the effect of changes in key gene expression on cellular glucose metabolism and senescence,and further explore its possible mechanism.Methods:1.P2-3MSCs(early passage,EP)and P9-10MSCs(late passage,LP)from 1-2months healthy male Wistar rats were obtained by whole bone marrow adherent method and serial expansion in vitro.Cellular senescence was evaluated by observing cellular morphology,detecting the activity of senescence associated?-galactosidase(SA-?-gal)and expression of senescence related factor P16^INK4A m RNA,and then replicative senescence model of MSCs in vitro was established.2.The gene expression profiles of young MSCs and senescent MSCs were detected by RNA Seq.According to the difference multiple and significance,significantly differentially expressed genes related to glucose metabolism were screened out,and the expression levels of related genes were verified by RT-q PCR.3.The expression of phosphofructokinase,muscle(PFKM)in MSCs was up-regulated by gene overexpression.Senescence indexes and glucose metabolism related indexes,including glucose consumption,lactate secretion level,and ATP content were detected,and the effects of PFKM overexpression on glucose metabolism and senescence of MSCs were investigated.4.Bioinformation website predicted that mi RNAs could combine with PFKM,and the expression levels of mi RNAs in EPMSCs and LPMSCs were verified by RT-q PCR.The targeted binding of mi R-141-3p to PFKM was verified by dual luciferase reporter gene assay.5.The effects of mi R-141-3p overexpression or inhibition on PFKM expression in MSCs were detected by RT-q PCR and Western Blot.The indexes related to senescence and glucose metabolism were detected.The molecular mechanism of mi R-141-3p/PFKM affecting the senescence of MSCs was further explored through rescue experiment.Results:1.LPMSCs changed significantly in morphology,such as increased cell areas,unclear boundary and decreased aspect ratio,when compared with EPMSCs.The positive ratio of SA-?-gal staining was elevated and the senescence-associated factors P16^INK4awas also obviously increased.In the following experiment,we used EPMSCs as young cells and LPMSCs as senescent cells.2.The results of RNA Seq showed that there were 3695 genes notably expressed,including 1050 up-regulated genes and 2645 down-regulated genes.The GO and KEGG Pathway enrichment analysis of differentially expressed genes showed that biological processes and pathways such as cell proliferation and differentiation were enriched in senescence MSCs.However,cell metabolism and other signaling pathways were significantly down-regulated in senescent MSCs.Subsequently,we screened and identified significantly heterogenous genes related to glucose metabolism,such as PFKM,PFKL and PKM.Compared with young MSCs,PFKM expression was most significantly decreased in senescent MSCs.3.After PFKM overexpression,senescent MSCs presented young morphology,and the positive ratio of SA-?-gal staining and expression level of P16^INK4a m RNA were decreased.Glucose consumption and lactate secretion increased,but ATP content decreased.4.Bioinformatics predicted that mi RNAs had potential complementary binding sites with PFKM m RNA 3'-UTR region.The three mi RNAs with the highest overall score were selected,including mi R-141-3p,mi R-542-3p and mi R-383.RT-q PCR results showed that the expression levels of the above three mi RNAs in senescent MSCs was notably higher than that in young cells.Furthermore,mi R-141-3p and mi R-542-3p,which are significantly up-regulated,were selected for overexpression.Because PFKM expression was down-regulated more strikingly after mi RNA-141-3p overexpression,mi R-141-3p was selected for subsequent experiments.5.Dual luciferase reporter gene assay showed that mi R-141-3p could bind to PFKM 3'-UTR,and PFKM was the direct target gene of mi R-141-3p.6.The overexpression of mi R-141-3p significantly down-regulated the glucose metabolism of young MSCs.The cell body became bigger and the shape became irregular.The positive ratio of SA-?-gal staining and expression level of P16^INK4am RNA increased.While mi R-141-3p inhibition up-regulated the glucose metabolism of senescent MSCs,and the positive rate of senescent cells and expression level of P16^INK4a m RNA were significantly reduced.7.Co-expression of PFKM and mi R-141-3p in senescent MSCs could down-regulate the higher expression levels of PFKM m RNA and protein caused by the PFKM overexpression,and attenuate the effect of PFKM overexpression to inhibit MSC senescence.Conclusion:1.PFKM is the most significantly differential glucose metabolism-related gene screened out in senescent MSCs.2.PFKM expression was decreased in senescent MSCs.Overexpression of PFKM can up-regulate glucose metabolism of senescent MSCs and thus inhibit cellular senescence.3.The expression of miR-141-3p is elevated in senescent MSCs,and PFKM is a direct target gene of mi R-141-3p.Mi R-141-3p can modulate the expression of PFKM to affect glucose metabolism and further regulate MSC senescence.