Study Of The Roles Of IncRNA-ANCR In Human Adipose Tissue-derived Stem Cells

BackgroundAdipose tissue-derived stem cell(ADSC)is multipotent with mesodermal origin and is isolated from adipose tissues.It has been the hot spot of the basic studies and applications of mesenchymal stem cell(MSC)for its abundant sources,convenient and easy to cultivate,multi-directional differentiation capacity,immunomodulatory properties,et al.ADSC can be used for repair and regeneration of various tissues and organs,such as skin,bone,cartilage,tendon,ligament,peripheral nerve and so on.The biological characteristics of ADSC,such as proliferation,multi-directional differentiation and migration,are the key factors that affect the beneficial effects of ADSC on tissue repair.MSC often exert their recovery function on damaged tissues in inflammatory microenvironment.Inflammatory cytokines will affect the regenerative and tissue repair functions of MSC by regulating the biological characteristics of MSC.Therefore,more and more attention has been paid to the changes and regulation of biological characteristics of ADSC under inflammatory microenvironment.Recent studies have shown that long noncoding RNA(IncRNA)plays an important role in regulating the complex gene expression networks of stem cells.IncRNA-ANCR/DANCR(Anti-differetiation Noncoding RNA,ANCR)is widely expressed in multiple human tissues and plays different regulatory roles in stem cells derived from different tissues.For example,it has been reported that ANCR can inhibit the epidermal differentiation,the osteogenic differentiation of human bone marrow-derived mesenchymal stem cell(BMSC)and the adipogenic,osteogenic and neural differentiation of dental tissue-derived stem cell(DTSC).On the other hand,ANCR was found to promote the proliferation and chondrogenic differentiation of synovial tissue-derived mesenchymal stem cell(SMSC).Nowadays the effects of ANCR on the biological characteristics of human ADSC(hADSC)have not been reported yet.Our pilot studies showed that dexamethasone(DEX),which is closely related to inflammation,could promote the expression of ANCR in hADSC,suggesting that ANCR may also be regulated by inflammatory factors in the inflammatory microenvironment.Therefore,after screening the inflammatory factors that affect the expression of ANCR,we applied DEX and TNF-? to hADSC and studied the effect of ANCR on the biological characteristics of hADSC as well as the underlying mechanisms.This study enriched the understanding of the mechanism related to the changes of biological characteristics of hADSC in inflammatory microenvironment,and provided the theoretical basis for the application of hADSC in tissue damage.Objective1.To investigate the regulatory effects of ANCR on the proliferation,cell cycle,apoptosis,migration,adipogenic and osteogenic differentiation capacities of hADSC.2.To investigate the effect of DEX on the expression of ANCR in hADSC and to explore the role of ANCR in the DEX-induced changes of biological characteristics of hADSC.3.To screen the inflammatory factors which regulate the expression of ANCR in hADSC,and to investigate the function of ANCR in the TNF-a-induced changes of biological characteristics of hADSC.4.To elucidate the mechanisms underlying the regulatory effects of TNF-a and DEX on the expression of ANCR in hADSC.Methods1.Effects of ANCR on the biological characteristics of hADSCSVF(Stromal Vascular Fraction)was obtained from abandoned adipose tissue after liposuction and hADSC was isolated by adherent culture of SVF.The expressions of MSC-specific surface markers in P3 hADSC were analyzed by flow cytometry.The letivirus-based ANCR knockdown and over-expression plasmids were constructed and used to knock-down and over-express ANCR in hADSC respectively.CCK-8 was used to assess the proliferation of hADSC.The cell cycle of hADSC was detected by Muse cell analyzer.The percentage of apoptotic cells in hADSC was evaluated after staining with Annexin-V and 7-AAD followed by flow cytometry using Muse cell analyzer.The in vitro scratch assay was used to detect the migration of hADSC.Oil red O staining was used to characterize the adipogenic differentiation capabilities of hADSC after 7 days of adipogenic induction.Alizarin red staining was used to assess the osteogenic differentiation capacities of hADSC after 14 days of osteogenic induction.In addition,real-time PCR analysis was used to quantify the expressions of the following genes in hADSC after knockdown and over-expression of ANCR respectively:proliferation-related genes C-FOS,C-JUN;cell cycle-regulated genes P53,P21 and CHEK1;apoptosis-related genes Caspase8,Caspase3,NF-?B and Bcl-2;chemokine receptors CXCR4 and CXCR7;transcription factors that play crucial roles in adipogenic induction(PPARy and C/EBPa),lipid synthase and osteogenic differentiation(RUNX2 and ALP).2.The effect of DEX on the biological characteristics of hADSC and the underlying functions of ANCRThe proliferation,cell cycle,apoptosis and migration of hADSC were studied after DEX treatment with three different concentrations(1×10-6mol/L,1×10-7mol/L,1×10-8 mol/L).Real-time PCR analysis was used to further characterize the expression of ANCR and genes mentioned in step 1 after DEX treatment.The proliferation,cell cycle and migration of ANCR-knockdown hADSC were further investigated after treatment with 1×10-7mol/L DEX and real-time PCR analysis was performed to evaluate the expression of genes mentioned above.3.The effect of TNF-a on the biological characteristics of hADSC and the underlying functions of ANCRSix pro-inflammatory factors(TNF-a,IL-1?,IFN-y,IL-6,IL-8,and MCP-1)and two anti-inflammatory factors(IL-13 and IL-10)were added to the hADSC culture individually and the expression of ANCR was assessed by real-time PCR analysis.The hADSC was treated with TNF-a at 10 ng/ml and 50 ng/ml respectively in vitro and the proliferation,cell cycle,apoptosis,migration,adipogenic and osteogenic differentiation abilities of hADSC were characterized followed by real-time PCR analysis for the expressions of genes related to proliferation,cycle,apoptosis,migration,adipogenesis and osteogenesis.After treatment with 10 ng/ml TNF-?,the expressions of genes that related to the proliferation,cell cycle and migration of ANCR-overexpression hADSC were further investigated by real-time PCR.4.The regulatory mechanisms of TNF-a and DEX in the expression of ANCR in hADSCThe cells treated with 10 ng/ml TNF-a were harvested at different time points and western blot was used to investigate the activation of NF-?B and mTOR signaling pathway in hADSC.Furthermore,the inhibitors of NF-?B pathway(BAY11-7028,DEX)and mTOR pathway(Rapamycin and PP242)were added to the hADSC culture respectively followed by 10 ng/ml TNF-a treatment,and western blot was performed to detect that whether the corresponding signaling pathway were inhibited.Finally,real-time PCR was used to quantify the expression of ANCR.Results1.Effects of ANCR on the Biological Characteristics of hADSCMore than 95%of P3 hADSC were positive for the expression of MSC surface markers CD44,CD73,CD90 and CD105 but negative for the expression of hematopoietic stem cell markers such as CD45.Knockdown of ANCR could promote the proliferation of hADSC,increase the proportion of cells in S phase,decrease the proportion of cells in G0/G1 phase,and promote the migration,adipogenic and osteogenic differentiation of hADSC.Overexpression of ANCR could inhibit the proliferation of hADSC,decrease the proportion of cells in S phase,increase the proportion of cells in G0/G1 phase,and inhibit the migration,adipogenic and osteogenic differentiation of hADSC.Knockdown and overexpression of ANCR had no significant effect on the percentage of apoptotic cells in hADSC.2.The effect of dexamethasone on the biological characteristics of hADSC and the underlying functions of ANCRDEX could increase the expression of ANCR in hADSC in a dose-dependent manner.High concentrations of DEX(1×10-6mol/L,1×10-7mol/L)could inhibit the proliferation of hADSC in vitro,decrease the proportion of cells in S phase and increase the proportion of cells in G0/G1 phase,inhibit the migration of hADSC,promote the expression of the apoptosis-related and adipogenesis-related genes PPARy and C/EBPa.Low concentration of DEX(1 × 10-8mol/L)promoted the expression of RUNX2 and ALP but had no significant effects on the proliferation,cell cycle,apoptosis and migration of hADSC.Knockdown of ANCR could partially reverse the simulative effect of 1×10-7mol/L DEX on the proliferation,cycle and migration of hADSC.3.The effect of TNF-a on the biological characteristics of hADSC and the underlying functions of ANCR.TNF-a,IL-1? and IFN-y could specifically reduce the expression of ANCR in hADSC.10 ng/ml and 50 ng/ml of TNF-a could promote the proliferation of hADSC,increase the proportion of cells in S phase,decrease the proportion of cells in G0/G1 phase,promote the migration of hADSC and inhibit the adipogenic and osteogenic differentiation of hADSC.TNF-a also could play an anti-apoptotic role by promoting the expression of anti-apoptotic factor NF-?B in hADSC.Over-expression of ANCR could weaken the promoting effect of TNF-a on the proliferation,cycle and migration of hADSC.4.The regulatory mechanisms of TNF-? and DEX in the expression of ANCR in hADSCTNF-a could activate NF-?B and mTOR signal pathways in hADSC.BAY 11-7082,DEX,Rapamycin and PP242 could specifically inhibit the NF-?B and mTOR signaling pathway.BAY 11-7082 and DEX could inhibit the TNF-a induced down-regulation of ANCR in hADSC.In addition,BAY11-7082 and DEX could specifically promote the expression of ANCR by inhibiting NF-?B signaling in hADSC while no significant difference on the expression of ANCR was observed after Rapamycin and PP242 treatment.Conclusions1.ANCR could regulate the proliferation,cell cycle,migration,adipogenic and osteogenic differentiation capabilities of hADSC.2.DEX could induce the expression of ANCR in hADSC in a dose dependent manner,and knockdown of ANCR could decrease the inhibitory effect of 1 × 10-7 mol/L DEX on the proliferation,cell cycle and migration of hADSC.These results suggested that ANCR may be involved in the high-concentration DEX-mediated regulation of proliferation,cell cycle and migration of hADSC.3.Pro-inflammatory cytokine TNF-a could reduce the expression of ANCR in hADSC,and overexpression of ANCR could reverse the promotive effect of TNF-a on the proliferation,cell cycle and migration of hADSC.These results suggested that ANCR might be involved in the TNF-a-mediated regulation of hADSC proliferation,cell cycle and migration.We speculated that the decrease of ANCR was closely related to the changes of the proliferation,cell cycle and migration of hADSC in the inflammatory microenvironment.4.The expression of ANCR in hADSC was regulated by NF-?B signaling.1 x 10-7 mol/L DEX could promote the expression of ANCR in hADSC by inhibiting the NF-?B pathway.Furthermore,1× 10-7 mol/L DEX could antagonize TNF-?-induced ANCR degradation by specifically inhibiting the TNF-?-induced activation of NF-?B signaling.