Telomere-Related Gene TINF2 Regulates Cellular Senescence And Osteogenic Differentiation In Mesenchymal Stem Cells By Mediating Mitochondrial Function Via NF-κB Pathway

Mesenchymal stem cells(MSCs)are undifferentiated cells with the capacity of self-renewal and multilineage differentiation.It has been proved that MSCs are closely concerned with the occurrence and progression of osteoporosis(OP).OP is an agerelated disease characterized by a reduction in the number of osteoblasts.Osteoblasts originate from bone marrow MSCs,so the pathogenesis of OP is mainly due to the decreased osteogenic differentiation of MSCs.It was shown that the density of bone minerals could be improved by inducing MSCs to differentiate into osteoblasts,thus preventing OP deterioration.However,MSCs become senescent with in vitro serial cultivation,and their osteogenic differentiation potentials are also attenuated,which severely restricts their basic research and clinical applications.Hence,it is of great significance to reveal the molecular mechanisms regulating stem cell senescence and osteogenic differentiation for improving stem cell function and osteoporosis treatment.Telomere attrition and mitochondrial dysfunction are the main hallmarks of aging,and there is a crosstalk regulation between them.Telomere shortening not only can give rise to cellular senescence and mitochondrial dysfunction,but also is related to the diminished differentiation potentials of stem cells.Reduced mitochondrial ROS production can also alleviate telomere attrition and DNA damage caused by cellular senescence.In addition,mitochondria are the main energy source for osteogenic differentiation of stem cells,and increased mitochondrial ATP synthesis contributes to the fracture healing.In our previous work,we found that the osteogenic differentiation in senescent MSCs was attenuated.Transcriptome sequencing results revealed that telomere-related genes in senescent MSCs were significantly down-regulated,especially TINF2,the core component of telomere shelterin complex.Thereby,we propose whether the decreased osteogenic differentiation in senescent MSCs is concerned with the down-regulated TINF2.Combined with KEGG Pathway enrichment analysis results,NF-κB signaling pathway in senescent MSCs was obviously lowered,and there was close relationship between NF-κB signaling pathway and stem cell senescence,osteogenesis and mitochondrial function.We thus speculate that telomere-related gene TINF2 might be involved in regulating MSC senescence and osteogenic differentiation by mediating mitochondrial function via NF-κB signaling pathways.Purpose:To screen out telomere-related differential genes with significant changes in senescent MSCs,clarify the impact of differentially expressed genes on the MSC senescence and osteogenic differentiation,and further explore the mechanism,so as to not only provide the new approaches and novel targets for delaying stem cell senescence and improving osteogenic differentiation,but also offer the experimental basis for clinical treatment of osteoporosis.Methods:1.Primary MSCs were extracted from healthy male Wistar rats(1～2 months old)by whole bone marrow adherent method.And MSCs(early passage MSCs,EPMSCs)and MSCs(late passage MSCs,LPMSCs)were obtained through in vitro serial subculture.Then cell morphology was observed,and age-related indexes such as cell surface area,cell length-width ratio,SA-β-gal activity and mRNA expression of agerelated factor pI6INK4a were detected to set up MSC replicative senescence model in vitro.2.Transcriptome sequencing was used to detect the gene expression profiles of young and senescent MSCs,and then telomere-related differentially expressed genes with significant changes were screened out.TRF1 and TRF2 interacting nuclear protein 2(TINF2)were determined to be the most remarkable differential gene.The expressio,levels of TINF2 were further verified by RT-qPCR and Western Blot.3.MSCs with TINF2 knock-down(sh-TINF2)or over-expression(Lv-TINF2)were obtained by lentivirus infection(TINF2 knock-down in young MSCs and overexpression of TINF2 in senescent MSCs),and their effects on MSC senescent and osteogenic differentiation were investigated.Age-related indexes including cell morphology,SA-β-gal activity,p16INK4a mRNA expression,and osteogenic differentiation ability(Alizarin red staining,expression of Runx2,a marker of osteogenic differentiation by Western Blot)were detected,respectively.Additionally,TINF2 was over-expressed in young MSCs by lentivirus infection to examine the alterations in cell senescence and osteogenic differentiation.4.The influence of TINF2 knock-down or over-expression in MSCs on telomeric and mitochondrial function was explored.Detection of telomeric function included telomere length and the expression of γH2AX,a marker of telomere injury).And ATP content and ROS levels were tested to figure out mitochondrial function.5.The expression levels of NF-κB signaling pathway after TINF2 knock-down or over-expression in MSCs were detected by Western Blot.Then NFκB signaling pathway was activated by the activator PMAin sh-TINF2 MSCs to explore its influence on the mitochondrial function and cellular senescence.Results:1.Compared with EPMSCs,LPMSCs displayed morphological alterations,such as cytosolic spreading and unclear cell boundaries.The cell length-width ratio was significantly reduced,while the surface area was extraordinarily increased.The positive rate of SA-β-gal was elevated,and the expression of p16INK4a mRNA was up-regulated.EPMSCs were thus used as young MSCs and LPMSCs as senescent MSCs in subsequent studies.2.Transcriptomic analysis indicated that there were 3188 differentially expressed genes(918 up-regulated genes and 2270 down-regulated genes)in senescent MSCs compared with young MSCs,among which 11 telomere-related differentially expressed genes had the most prominent changes.GO and KEGG Pathway enrichment analyses showed that telomere maintenance,site of double-strand break and telomerase RNA binding were weakened in senescent MSCs,and NF-κB signaling pathway,apoptosis and non-homologous end-j oining were down-regulated in senescent MSCs.Among the 11 telomere-related differentially expressed genes,the alterations of Parp and TINF2 were most observable.Since TINF2 is the core component of telomere complex and plays an essential role in maintaining telomeric function,TINF2 was subsequently used as research target.Consistent with the results of transcriptome sequencing,RT-qPCR and Western blot further confirmed that TINF2 was significantly down-regulated in senescent MSCs.3.TINF2 knock-down made young MSCs present senescent morphological characteristics.The cell surface area increased,while the cell length-width ratio decreased.The positive rate of SA-β-gal and the expression of p16INK4a mRNA were both elevated.Meanwhile,the calcium deposition in bone matrix was diminished,and the protein levels of Runx2 was reduced.After TINF2 over-expression,the senescent MSCs changed from cytosolic spreading and irregular to long spindles with clear boundaries.The cell surface area decreased and the length-width ratio increased.Both the positive rate of SA-β-gal and the expression of p16INK4a mRNA were lower,together with enhanced calcium deposition in bone matrix and Runx2 protein expression.Furthermore,over-expressed TINF2 in young MSCs had no significant effect on cellular senescence,but remarkably strengthened osteogenic differentiation.4.After knock-down of TINF2,the telomere length of MSCs was shortened,and the expression of telomere damage marker yH2AX was enhanced.In the meantime,mitochondrial ROS level was increased while ATP content was reduced.After TINF2 over-expression,the telomere length of MSCs was prolonged,and the expression of telomere damage marker yH2AX was reduced,accompanied by reduced mitochondrial ROS level and increased ATP content.The above results suggest that TINF2 knockdown can attenuate telomeric and mitochondrial function of MSCs,while TINF2 overexpression can enhance telomeric and mitochondrial function of MSCs.5.After TINF2 knock-down,the protein expression levels of p-NF-κB p65 were markedly down-regulated,while there was no significant change in the expression of NF-κB p65.After TINF2 over-expression,the protein expression levels of p-NF-κB p65 were significantly up-regulated,while the expression levels of NF-κB p65 were unchanged.It was suggested that TINF2 knock-down can restrain NF-κB signaling pathway,while over-expressed TINF2 can activate NF-κB signaling pathway.Moreover,with the treatment of PMA,the activator of NF-κB signaling pathway,intracellular ROS level in sh-TINF2 MSCs was significantly decreased,while ATP content was remarkably increased.The senescent MSCs changed from cytosolic spreading and irregular to long spindles with clear boundaries.The cell surface area was reduced and the length-width ratio was elevated.Both the positive rate of SA-βgal and the expression of p16INK4a mRNA were lower.The results indicate that the activation of NF-κB signaling pathway can notably ameliorate the declined mitochondrial function and delay MSC senescence caused by TINF2 knock-down.Conclusion:1.In senescent MSCs,the expression of telomere-related gene TINF2 is decreased.2.TINF2 knock-down promotes MSC senescence and inhibits the osteogenic differentiation.TINF2 over-expression ameliorates MSC senescence and enhances the osteogenic differentiation.3.TINF2 regulates cellular senescence and osteogenic differentiation in MSCs by mediating telomeric and mitochondrial function via NF-κB pathway.