An Experimental Study On Long Non-coding RNA TCONS₀0041960 Inhibits Adipogenesis And Enhances Osteogenesis Of Steroid-induced Rat BMSCs

BackgroundOsteonecrosis of the femoral head?ONFH?has become a common clinical disease.In China,it's estimated that 8 million people are suffering from ONFH.Especially extensive clinical use of steriod has been identified as the main risk factor for non-traumatic ONFH,frequently occurring in young adults leading to bilateral ONFH,loss of function and even disability,which severely reduces the patient's quality of life.Presently,there is still no exact and effective strategy to prevent the the occurrence of steroid-induced ONFH,eventually had to resort to surgery operation-total hip arthroplasty,meanwhile this unquestionably increases the burden on patients' family,uncertain long-term effect likewise increases the economic burden of the society.To date,the development of science and technology don't help people reach an agreement on understanding of the pathogenesis of steroid-induced osteonecrosis.Therefore,further study of the molecular mechanism of steroid-induced ONFH and exploration of targeted therapy have important scientific significance and application prospects.In the field of life sciences,non-coding RNAs,known as the "dark matter" of the life world,has been increasingly found to be involved in diverse biological processes such as cell cycle regulation,embryonic development,and cell differentiation.It's reported that miRNAs and lncRNAs have an important role in regulation of BMSCs abnormal differentiation?osteogenesis and adipogenesis?.BMSCs abnormal differentiation is the key link during the startup and development of ONFH.For steroid-induced ONFH prevention,it is also crucial to promote osteogenesis,especially enhance the expression of osteogenic key gene Runx2 except to inhibiting adipogenic key gene PPARy of BMSCs.As we known,the occurrence of ONFH is the result of multiple genes regulation and multiple factors impact.There is network regulation between different molecules,such as a lncRNA or miRNA can control multiple target genes,and multiple lncRNAs or miRNAs can regulate the mutual target genes at the same time,there may be mutual regulatory relationship between lncRNAs and miRNAs or bewteen lncRNAs themselves.At present,there have been no reports in domestic and foreign literatures that lncRNA-miRNA-mRNA multi-target network regulating rat BMSCs abnormal differentiation for prevention steroid-induced ONFH.Therefore,this study was aiming at examing lncRNA-miRNA-mRNA multi-target network regulation to explore development and prevention method of ONFH,which has important scientific significance and value.PurposeUse gene chip and bioinformatics analysis to select differentially expressed lncRNAs and miRNAs in steroid-induced BMSCs by comparison with normal BMSCs,verify the target lncRNA interaction with miRNAland miRNA2,meanwhile verify GILZ is a target gene of miRNAl,Runx2 is a target gene of miRNA2.By up-regulation of target IncRNA expression lead to down-regulation of miRNA1 and miRNA2,ultimately repression of PPAR? expression and promotion of Runx2 expression,which is double efficiency that suppression of adipogenesis and strengthen osteogenesis.To further investigate the advantages and mechanism of lncRNA-miRNA-mRNA multi-target network regulating BMSCs abnormal differentiation for prevention steroid-induced ONFH.This study was divided into five parts as followed:Part lAnalysis differentially expressed lncRNAs and miRNAs in steroid-induced BMSCsMethod?1?Make BMSCs:The primary BMSCs were extracted from the tibia and femur of rats and cultured to P3 generation in vitro.Then the surface antigen CD29,CD34,CD45 and CD 105 were identified by flow cytometry.?2?Steriod induced BMSCs differentiation:BMSCs were induced with 10-6M dexamethasone;After 7 days of induction,the Oil red"0" assay was used to confirm the success of steriod induction.?3?Differentially expressed lncRNAs and miRNAs were selectively in steroid-induced BMSCs:the differences of lncRNAs and miRNAs between normal BMSCs and steroid-induced BMSCs were detected by gene chip.?4?Verify the results of gene chip and the target genes:we randomly selected 4 lncRNAs and 4 miRNAs,through qRT-PCR exam the expression of target lncRNA and miRNA in steroid-induced BMSCs,the expression of PPARy,GILZ and Runx2.?5?SPSS 17.0 software was used for data processing and statistical analysis.Results?1?CD29 and CD105 expression of P3 generation BMSCs were 99.76%and 99.42%,respectively.CD34 and CD45 were negative,and the positive rate was merely 1.23%,1.41%,the high purity BMSCs were obtained.?2?With 10-6M dexamethasone induced BMSCs 7 days,a large number of lipid droplets appeared in the Model group,no lipid droplets were found in the Control group through Oil red "0" assay,indicating that the steroid-induced cell model was set successful.?3?Compared Model group with Control group,10 miRNAs were up-regulated and 16 miRNAs were down-regulated.79 lncRNAs were up-regulated and 72 lncRNAs were down-regulated.?4?qRT-PCR results showed that steroid-induced Model group compared with Control group,lncRNA TCONS₀0041960 expression level significantly was lowered?P<0.01?,IncRNA TCONS₀0083120 expression level significantly was enhanced?P<0.05?,miR-204-5p and miR-125a-3p expression level were increased obviously?P<0.05?,miR-146a-5p and miR-301a-3p expression level were decreased?P<0.05?,and adipogenic gene PPARy expression level was elevated?P<0.05?,but GILZ and Runx2 gene expression levels were decreased?P<0.05?.Part 2 The impact of IncRNA TCONS₀0041960 up-regulation on steroid-induced BMSCs differentiationMethod?1?Construction of overexpressed IncRNA TCONS₀0041960?Ex-Lnc?recombinant lentivirus vector,negative control oligonucleotides IncRNA TCONS₀0041960?Ex-NC?;build overexpressed IncRNA TCONS₀0041960 in BMSCs.?2?The study was divided into 4 groups:Blank group,Dex group,Ex-Lnc+Dex group,Ex-NC+Dex group.?3?Detected lncRNA TCONS₀0041960 expression in Ex-Lnc+Dex group and Ex-NC+Dex group by qRT-PCR to see if recombinant lentivirus vector was successful;After 14 days of steriod-induction,respectively examed gene expression of osteogenic genes Runx2,Osterix and Osteocalcin,gene expression of adipogenic genes PPARt?,C/EBP? and GILZ by qRT-PCR;respectively detected protein expression of osteogenic genes Runx2,Osterix and Osteocalcin,protein expression of adipogenic genes PPARy,C/EBPo? and GILZ by Western blot.At the same time,test the content of triglyceride?TG?and alkaline phosphatase?ALP?activity,and the Oil red "O" was dyed.?4?SPSS 17.0 software was used for data processing and statistical analysis.Results?1?Successfully construction of overexpressed IncRNA TCONS₀0041960?Ex-Lnc?recombinant lentivirus vector,negative control oligonucleotides IncRNA TCONS₀0041960?Ex-NC?,the lentivirus titers were 1×108TU/ml and 5×108TU/ml respectively.Successfully established overexpressed lncRNA TCONS₀0041960 in BMSCs,MOI?multiplicity of infection?=2.?2?Comparison with Dex group and Ex-NC+Dex group,lncRNA TCONS₀0041960 expression in Ex-Lnc+Dex group was up-regulated significantly?P<0.01?,which indicated that recombinant lentivirus vector successfully infect BMSCs.?3?In Ex-Lnc+Dex group,gene and protein expression of osteogenic genes Runx2,Osterix and Osteocalcin were higher than Dex group and Ex-NC+Dex group?P<0.05?;however,gene and protein expression of adipogenic genes PPARy,C/EBP? were lower than Dex group and Ex-NC+Dex group?P<0.05?,but GILZ?PPAR? Inhibitor?was higher than Dex group and Ex-NC+Dex group?P<0.05?.?4?The Oil red "O" results showed that the lipid droplets in Ex-Lnc+Dex group were less than Dex group and Ex-NC+Dex group,and then the content of triglyceride?TG?in Ex-Lnc+Dex group was lower than Dex group and Ex-NC+Dex group?P<0.05?,however,alkaline phosphatase?ALP?activity of Ex-Lnc+Dex group was higher than Dex group and Ex-NC+Dex group?P<0.05?.Part 3 The impact of miR-204-5p and miR-125a-3p down-regulation on steroid-induced BMSCs differentiationMethod?1?miR-204-5p Inhibitor and miR-125a-3p Inhibitor,miR-204-5p negative control and miR-125a-3p negative control were synthetic,and liposomal transfection was used to transfer them into BMSCs.?2?The study was divided into 5 groups:Blank group,Dex group,NC+Dex group,miR-204-5p Inhibitor+Dex group,miR-125a-3p Inhibitor+Dex group.?3?Detect miR-204-5p and miR-125a-3p expression in five groups by qRT-PCR to see if transfection was successful;After 14 days of steriod-induction,respectively exam gene expression of osteogenic genes Runx2,Osterix and Osteocalcin,gene expression of adipogenic genes PPAR?,C/EBP? and GILZ by qRT-PCR;respectively detect protein expression of osteogenic genes Runx2,Osterix and Osteocalcin,protein expression of adipogenic genes PPAR?,C/EBP? and GILZ by Western blot.At the same time,test the content of triglyceride?TG?and alkaline phosphatase?ALP?activity,and the Oil red "O" was dyed.?4?SPSS 17.0 software was used for data processing and statistical analysis.Results?1?qRT-PCR results showed that miR-204-5p expression and miR-125a-3p expression are lower than Dex group and NC+Dex group respectively,which showed that transfection successfully?P<0.05?.?2?In miR-204-5p Inhibitor group,gene and protein expression of osteogenic genes Runx2,Osterix and Osteocalcin are higher than Dex group and NC+Dex group?P<0.05?;In miR-125a-3p Inhibitor group,gene and protein expression of adipogenic genes PPAR?,C/EBP? are lower than Dex group and NC+Dex group?P<0.05?,but GILZ?PPARy Inhibitor?is higher than Dex group and NC+Dex group?P<0.05?.?3?The Oil red "O" results showed that the lipid droplets in miR-125a-3p Inhibitor group are less than Dex group and NC+Dex group,and then the content of triglyceride?TG?in miR-125a-3p Inhibitor group is lower than Dex group and NC+Dex group?P<0.05?,however,alkaline phosphatase?ALP?activity of miR-204-5p Inhibitor group is higher than Dex group and NC+Dex group?P<0.05?.?4?SPSS 17.0 software was used for data processing and statistical analysis.Part 4 Long non-coding RNA TCONS₀0041960 enhances osteogenesis and inhibits adipogenesis of steroid-induced rat BMSCs by targeting miR-204-5p and miR-125a-3pMethod?1?The study was divided into 5 groups:Blank group,Dex group,Ex-Lnc+Dex group,miR-204-5p Inhibitor+Dex group,miR-125a-3p Inhibitor+Dex group.After 14 days of steriod-induction,respectively exam gene expression of osteogenic genes Runx2?Osterix and Osteocalcin,gene expression of adipogenic genes PPAR?,C/EBP? and GILZ by qRT-PCR.?2?miR-204-5p mimic and miR-125a-3p mimic were synthetic,and liposomal transfection was used to transfer them into BMSCs.?3?Test the content of triglyceride?TG?in Blank group,Dex group,Ex-Lnc+Dex group,miR-125a-3p mimic group,Ex-Lnc+miR-125a-3p mimc+Dex group and alkaline phosphatase?ALP?activity in Blank group,Dex group,Ex-Lnc+Dex group,miR-204-5p mimc+Dex,Ex-Lnc+miR-204-5p mimc+Dex group.Results?1?In miR-204-5p Inhibitor group and Ex-Lnc+Dex group,gene expression of osteogenic genes Runx2,Osterix and Osteocalcin are higher than Dex group?P<0.05?;In miR-204-5p Inhibitor group and Ex-Lnc+Dex group,gene expression of adipogenic genes PPAR'y,C/EBPaare lower than Dex group?P<0.05?,but GILZ?PPARy Inhibitor?is higher than Dex group?P<0.05?.These results showed that up-regulation lncRNA TCONS₀0041960 had the same effect with down-regulation miR-204-5p or miR-125a-3p?2?qRT-PCR results showed that miR-204-5p expression and miR-125a-3p expression are higher than Dex group and NC+Dex?P<0.01?.?3?ALP activity results indicated that increased ALP activity was also observed in the Ex-Lnc group compared with the Dex group?P<0.05?,but was reduced in the miR₂04-5p mimic group?P<0.05?,furthermore,an decreased ALP activity was observed in Ex-Lnc+miR-204-5p mimic+Dex group?P<0.05?;The content of triglyceride?TG?results showed that TG contents were reduced in the Ex-Lnc group compared with the Dex group?P<0.05?,but increased in the miR-125a-3p mimic group?P<0.05?,however,TG contents were elevated in Ex-Lnc+miR-125a-3p mimic+Dex group?P<0.05?.These results showd that lncRNA TCONS₀0041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR-204-5p and miR-125a-3p.Part 5 A preliminary study on the mechanism of IncRNA TCONS₀0041960 enhances osteogenesis and inhibits adipogenesis of steroid-induced rat BMSCs by targeting miR-204-5p and miR-125a-3p.Method?1?Use TargetScan and miRanda's website for bioinformatics analysis,predicted the potential interaction between IncRNA TCONS₀0041960 and miRNAs?miR-204-5p and miR-125a-3p?,target genes of miR-204-5p and miR-125a-3p.?2?Constructed IncRNA TCONS₀0041960 wild-type luciferase double report carrier?pmirGLO-TCONS₀0041960-WT?and luciferase dual report carrier?pmirGLO-TCONS₀0041960-MUT?respectively,and transfected miR-204-5p mimic and miR-204-5p NC into HEK 293Tcells,and transfected miR-125a-3p mimic and miR-125a-3p NC into HEK 293T cells,with dual luciferase report confirmed TCONS₀0041960 interaction with miR-204-5p or miR-125a-3p.?3?By RNA immunoprecipitation?RIP?dealing with BMSCs,used A/G magnetic beads to formation RNA immune co-precipitation complex combined with Ago2 antibody?experimental group?and RNA immune co-precipitation complex combined with negative control IgG antibody?control group?,further separated containing IncRNA TCONS₀0041960,miR-204-5p and miR-125a-3p RNA compounds,after purified RNA,through the qRT-PCR to detect IncRNA TCONS₀0041960,miR-204-5p and miR-125a-3p expression levels in the experimental group and the control group.?4?The biosensor chips containing IncRNA TCONS₀0041960-WT and IncRNA TCONS₀0041960-MUT were prepared respectively and through surface plasmon resonance technology?SPR?to detect response value between lncRNA TCONS₀0041960-WT with miR-204-5p mimic and miR-204-5p negative control at different time;response value between IncRNA TCONS₀0041960-WT with miR-125a-3p mimic and miR-125a-3p negative control at different time;response value between IncRNA TCONS₀0041960-MUT with miR-204-5p mimic and miR-204-5p negative control at different time;response value between lncRNA TCONS₀0041960-MUT with miR-125a-3p mimic and miR-125a-3p negative control at different time.?5?Study miRNAs?miR-204-5p or miR-125a-3p?impact on lncRNA TCONS₀0041960:the study was divided into 5 groups:miR-NC group,miR-204-5p mimic group,miR-125a-3p mimic group,miR-204-5p Inhibitor group,miR-125a-3p Inhibitor group.By qRT-PCR detected lncRNA TCONS₀0041960 expression level.?6?Study lncRNA TCONS₀0041960 impact on miR-204-5p or miR-125a-3p:the study was divided irnto 4 groups:Ex-NC group,Ex-Lnc group,si-NC group,si-Lnc group.By qRT-PCR detected miR-204-5p or miR-125a-3p expression level.?7?Constructed luciferase double report carrier pmirGLO-WT 3'UTR-Runx2 and pmirGLO-MUT 3'UTR-Runx2,co-transfected miR-204-5p mimic and miR-204-5p NC into HEK293T cells;Constructed luciferase double report carrier pmirGLO-WT 3'UTR-GILZ and pmirGLO-MUT 3,UTR-GILZ,co-transfected miR-miR-125a-3p mimic and miR-125a-3p NC into HEK293T cells.By dual luciferase report confirmed that Runx2 is target gene of miR-204-5p,GILZ is target gene of miR-125a-3p.By Western blot detect Runx2 and GILZ protein expression level in BMSCs.Results?1?TargetScan and miRanda bioinformatics analysis showed that TCONS₀0041960 contains sequences that recognize the seed regions of miR-204-5p and miR-125a-3p;the recognition sequences of miR-204-5p and miR-125a-3p matched the 3'-untranslated region sequences of Runx2 and GILZ respectively.?2?Dual luciferase report showed that lncRNA TCONS₀0041960 interacts with miR-204-5p and miR-125a-3p;Runx2 and GILZ are direct targets of miR-204-5p and miR-125a-5p,respectively.?3?RNA immunoprecipitation?RIP?assay results showed that lncRNA TCONS₀0041960?miR-204-5p and miR-125a-3p expression level were higher in Ago2 compounds than in IgG compounds?P<0.01?.?4?The surface plasmon resonance?SPR?assay results indicated that positive binding between wild-type TCONS₀004196 and miR-204-5p mimic,but not between MUT and TCONS₀004196 and miR-204-5p.In addition,positive binding was observed in the group co-transfected with wild-type TCONS₀004196 and miR-125a-3p mimic compared with the other three group.?5?qRT-PCR results revealed that compared with miR-NC group,lncRNA TCONS₀0041960 expression in miR-204-5p mimic group and miR-125a-3p mimic group was decreased?P<0.05?,lncRNA TCONS₀0041960 expression in miR-204-5p Inhibitor group and miR-125a-3p Inhibitor group was elevated?P<0.05?,which indicated that miRNAs?miR-204-5p and miR-125a-3p?could regulate lncRNA TCONS₀0041960 expression.?6?qRT-PCR results revealed that compared Ex-Lnc group with Ex-NC group,miR-204-5p and miR-125a-3p expression were both decreased?P<0.01?;compared si-Lnc group with si-NC group,miR-204₅p and miR-125a-3p expression were both elevated?P<0.05?,which showed that lncRNA TCONS₀0041960 could affect miR-204-5p and miR-125a-3p expression.?7?Western blot results showed that compared with Blank group and miR negative control,Runx2 protein expression level in miR-204-5p group was decreased;GILZ protein expression level in miR-125a-3p was reduced.Conclusion1.During steriod-induced BMSCs abnormal differentiation,the difference expression is more than 2 times,10 miRNAs were up-regulated and 16 miRNAs were down-regulated;79 lncRNAs were up-regulated and 72 lncRNAs were down-regulated.2.LncRNA TCONS₀0041960 up-regulation or miR-204-5p and miR-125a-3p down-regulation could effectively suppress adipogenesis meanwhile promote osteogenensis in steriod-induced BMSCs differentiation.3.LncRNA TCONS₀0041960 regulated the expression of Runx2 by negative regulation of miR-204-5p,and negatively regulated the expression of miR-125a-3p to regulate GILZ,thereby prevent adipogenic differentiation and actively promote the differentiation of ostegenic in BMSCs.4.LncRNA-miRNA-mRNA multi-target network regulating BMSCs abnormal differentiation is expected to be a new target for prevention and treatment ONFH.