| BackgroundBone marrow mesenchymal stem cells(BMSCs)have the capacity to differentiate into different cell tissues including osteoblast,adipocytes,chondrocytes as well as myocytes.BMSCs are not only found in bone marrow but also exist outside the bone tissue including fat and muscle tissue.When stimulated by appropriate signals,BMCs can drift away from their location,mostly bone tissue,to reach the target organ and this biological behavior is called "homing".In the presence of appropriate biological and physical factors with adequate specific signaling,BMSCs are able to differentiate into the required specific cell tissue.Additionally,they could divide and proliferate for 38+4 times.Because of these properties,BMSCs have become the main cell source for musculoskeletal tissue regeneration therapy studies.Bone tissue is a downstream product of osteoblast differentiation of BMSCs.At its basic level,osteoblast differentiation of MSCs is induced by expression of a key transcription factor,Runt-related transcription factor 2(Runx2).Runx2 activates and regulates osteogenesis as a target gene for many signaling pathways including,but not limited to,transforming growth factor-beta(T GF-b1),Bone morphogenetic protein(BMP),Wingless typel(Wnt),Hedgehog(HH),and(Nel)-like protein type 1(NELL-1).Following successful induction,downstream progression through pre-osteoblast differentiation into osteoblast,osteoblast maturation and matrix mineralization is regulated by other transcription factors including Osterix,BSP,OCN and OPN,This process is also affected by many other external factors,both physical and biological,most of which have been extensively studied.Chronic hyperglycemia,a characteristic feature of a Diabetic Mellitus,has been reported to negatively affect bone metabolism,thus thought to be predisposing affected individuals to the development of metabolic bone diseases like Osteoporosis.Recent in vitro studies have demonstrated that high glucose lowers the expression of the key osteoblast transcription factor Runx2,lowers intra-cellular BMP-2 and promotes osteoclast proliferation leading to decreased bone formation and increased bone resorption.Methods to enhance osteogenic differentiation of MSCs under high glucose are therefore needed to improve the efficacy of stem cell based musculoskeletal tissue regeneration therapy in diabetic patients.Nuclear receptor co-repressor(NCoR),a known transcriptional co-regulatory protein,is known to exert various repressing functions through recruitment of histone deacetylase(HDAC)and by interacting with numerous other transcription factors,including NF-Eb,AKT,and PPARy.Jin Z reported that HDAC9 inhibits PPARy activity in synergy with silencing mediator of retinoic acid and thyroid hormone receptors(SMRT)/NCoR co-repressors and identified HDAC9 as an important and physiologically relevant modulator of bone remodeling and skeletal homeostasis.Recently,Yi Qin demonstrated that NCoR negatively modulates osteogenic differentiation of rat mesenchymal stem cells through P13K/AKT cell signaling pathway.P13K/AKT is involved in glucose metabolism.Another previous study showed that NCoR knockdown promoted tissue insulin sensitivity in obese diabetic mice,implying that it could be involved in glucose metabolism.So,what is the effect of NCoR knock down on osteogenic differentiation of BMSCs under a high glucose microenvironment?PurposeIn this study,we aimed to observe the effects of NCoRsiRNA on proliferation and osteogenic differentiation of BMSCs isolated from Rat femora under high glucose microenvironment.Methods1.Expression plasmids for NCoRsiRNA and a negative non-target siRNA were designed according to the NCoR sequence from Gen Bank(EU006039.1).For transient transfections,cells were cultured with serum-free osteogenic medium for 2 days,in osteogenic medium for 2 days then transfected with 6 ?g of NCoRsiRNA construct or non-target siRNA for 2 d using HiPerFect(Qiagen)according to the manufacturer's protocols.The efficiency of NCoRsiRNA was then determined by western blot analysis.2.Effect of NCoRsiRNA on the proliferation of BMSCs under high glucose.In brief,isolated and identified rat MSCs were plated at a density of 1 x 10 3cells/well in 96-well plates for 24h.After attaining a confluence of 65%,Cells samples were transfected with NCoRsiRNA or non-target siRNA then cultured in osteogenic media containing four glucose concentrations(5.5,16.5,25 and 35mmol/L).After incubation for nine days,cell proliferation was determined using methyl thiazolyl tetrazolium(MTT)and absorbance determined at 490nm.3.Effect of NCoRsiRNA on osteogenic differentiation of rat BMSCs under high glucose.Cells transfected with NCoRsiRNA or non-target NCoRsiRNA were cultured in osteogenic media containing 25mmol/L(high glucose)and 5.5mmol/L glucose(control)respectively for 21 days.Osteogenic differentiation was determined by measuring quantitative changes in cellular ALP activity and calcium deposition.For confirmation,expression changes of osteoblast related genes;Run2,Osterix,OCN,OPN and BSP were determined by real time RT-PCR.Results1.Efficacy of NCoRsiRNA.Western blot analysis results showed that,compared with the control and non-target control,NCoRsiRNA transfected cells had significant decrease in NCoR gene expression after 2 days of transfection.2.NCoRsiRNA down regulated cell proliferation of BMSCs.BMSCs transfected with NCoRsiRNA or non-target NCoRsiRNA were cultured in osteogenic media containing four different glucose concentrations(5.5,16.5,25 and 35mmol/L).Compared with the control and negative control,transfection with NCoRsiRNA resulted in slight decrease in mesenchymal stem cell proliferation at all glucose concentrations.3.NCoRsiRNA promoted osteogenic differentiation of BMSCs under high glucoseObtained results showed a significantly higher ALP activity and calcium sedimentation in cells treated with NCoRsiRNA compared to those transfected with non-target NCoRsiRNA,as determined by ALP-ELISA kit and calcium essay diagnostic kit respectively.This was accompanied by significant increase in Runx2,Osterix,BSP,OCN and OPN gene expression,denoting that NCoRsiRNA enhanced osteogenic differentiation of rat BMSC under high glucoseConclusionPreventive regeneration therapy,through modulation of BMSC osteogenic differentiation under deranged microenvironments,may just be the ultimate solution to the poor prognosis of bone pathologies in diabetic patients.Our results showed that NCoR knockdown with NCoRsiRNA resulted in a significant and sustained increase in osteogenic differentiation of rat BMSCSs under high glucose.Although this was associated with reduced cell proliferation,this effect was insignificant(P<0.01).This could be used as an alternative to counteract the negative effects high glucose on osteogenic differentiation of BMSCs in diabetic patients consequently help combating the scourge of hyperglycemic induced bone pathologies in this special population.Combined with earlier reports that NCoR knock down promoted tissue insulin sensitivity,it would not only be used as a preventive regeneration therapy,but would also act as an adjunct to optimize insulin therapy in diabetic patients by increasing peripheral tissue insulin sensitivity. |
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