The Effects And Mechanisms Of SOX4 On Senescence In Mesenchymal Stem Cells

Background and objective: Mesenchymal stem cells(MSCs)are pluripotent stem cells with immunomodulatory function and pluripotent differentiation potential.At present,MSCs have been used in clinical treatment of various diseases and have shown good efficacy.However,the replicative senescence caused by expansion in vitro hinders the further optimization of the treatment strategy of MSCs.Our previous studies showed that the nuclear transcription factor SOX4 plays an important role in regulating the replicative senescent process of fibroblasts.Therefore,this study aims to explore the effect and potential mechanism of SOX4 on the senescence of MSCs,in order to provide new ideas for improving the clinical treatment effect of MSCs.Methods: MSCs with early passage(3rd generation,P3),late passage(8th generation,P8),SOX4 gene interference or overexpression,H2O2 pretreatment,mitochondrial reactive oxygen species(mtROS)clearance or lactic acid stimulation,cell senescence was assessed by β-galactosidase staining;Gene expression levels of SOX4,cell cycle suppressor p53,p21 and p16,senescence-associated secretory phenotype(SASP)factors IL-6,IL-1β and TNF-α,function related indicators IDO,COX-2,TGF-β,HLAG and HGF,mitochondrial dynamic molecules Drp1,Mff,Mid49,Mid51,Fis1,OPA1,Mfn1 and Mfn2 were detected by RT-q PCR;The protein expression levels of SOX4 and cell cycle inhibitors p53,p21 and p16 in MSCs were determined by western blot;The proliferation level of MSCs was detected by CCK8 assay;Surface markers,mtROS lactate dehydrogenase(LDH)and mitochondrial membrane potential levels of MSCs were determined by flow cytometry(FCM);Lactate content in culture supernatant was detected by lactate detection kit;Oil red O staining was used to examine the adipogenic potential of MSCs,and alizarin red staining was used to examine the osteogenic potential of MSCs;In addition,FCM was employed to detect the proliferation and activation levels of CD4+ T cells after co-culturing MSCs with human peripheral blood mononuclear cells(PBMCs)with different treatments.Results:(1)MSCs showed replicative senescence during long-term cultivation: Compared with MSC-P3,MSC-P8 showed increased volume and more granular material within the cytoplasm;The number of senescence-associated β-galactosidase(SA-β-gal)positive cells increased,the expression of p53,p21 and p16 genes and proteins increased,and the cell proliferation ability decreased;The expression of surface markers was elevated;The lipogenic potential decreased and osteogenic potential increased;The expressions of IDO,COX-2,TGF-β,HLA-G and HGF genes related to function were significantly decreased,while the expressions of IL-6,IL-1β and TNF-α genes of SASP were significantly increased;It manifested by a decreased ability to inhibit T cell proliferation and activation.(2)Downregulation of SOX4 induced MSCs senescence: Compared with MSC-P3,the SOX4 gene and protein expression of MSC-P8 were significantly decreased.After interference with SOX4 gene of MSC-P3,the senescence state of MSCs cells was aggravated,and the expression level of senescence related indexes was significantly increased;The surface marks had no change;Lipogenesis potential decreased;The gene expression of function related index decreased significantly,while the gene expression of SASP factor increased significantly;Decreased ability to inhibit T cell proliferation and activation.After overexpression of SOX4 gene of MSC-P8,the senescence state of MSCs cells was improved,and the expression level of senescence-related indicators was significantly decreased;The expression of surface marks decreased;Lipogenesis potential increased;The expression level of functional index gene was significantly increased;The ability to inhibit T cell proliferation and activation was enhanced.(3)Downregulation of SOX4 promotes mtROS generation by MSCs: Compared with MSC-P3,MSC-P8 showed significantly higher levels of mtROS,significantly lower mitochondrial membrane potential,and unchanged gene expression levels of mitochondrial dynamics molecules;After interfering with the SOX4 gene of MSCP3,MSCs showed significantly increased levels of mtROS,no obvious change in mitochondrial membrane potential,and no obvious change in mitochondrial dynamics molecules;After overexpression of the SOX4 gene of MSC-P8,MSCs showed significantly lower levels of mtROS,no obvious change in mitochondrial membrane potential,and no obvious change in mitochondrial dynamics molecules.(4)H2O2 treatment promoted MSCs senescence via promoting mtROS generation,and scavenging mtROS alleviated MSCs senescence: After H2O2 treatment of MSC-P3,the levels of mtROS in MSCs were obviously increased,and the state of cell senescence was aggravated,and the expression of senescence related indicators was obviously increased;The expression of functionally related index genes was significantly lower,and the expression of SASP index genes was significantly higher;It showed decreased ability to suppress T cell proliferation and activation.After scavenging mtROS of MSC-P8,the senescent state of MSCs was improved,and the expression of senescence related indicators was significantly decreased;The expression of functionally related index genes was significantly higher,and the expression of SASP index genes was significantly lower;It showed enhanced ability to suppress T cell proliferation and activation.(5)MSCs senescence induced by lactate may be related to downregulation of SOX4: Compared with MSC-P3,the expression of lactate dehydrogenase and the secretion of lactate increased in MSC-P8;After lactate stimulation of MSC-P3,the expression of SOX4 gene and protein was significantly decreased;The state of cellular senescence was aggravated,and the expression of senescence related indicators was obviously elevated;It showed reduced cell proliferation capacity.Conclusion:(1)Downregulation of SOX4 induced MSCs senescence;(2)SOX4 knockdown induced MSCs senescence via promoting mtROS generation;(3)MSCs senescence induced by lactate may be related to downregulation of SOX4.