| Part Ⅰ: Expression patterns of H19 and DNMTs in undifferentiated and osteogenic/adipogenic-differentiated BMSCs from patients with SONFHObjective: To detect the expression patterns of lnc RNA H19(H19)and DNA methyltransferases(DNMTs)in undifferentiated and osteogenic/adipogenic-differentiated bone marrow-derived mesenchymal stem cells(BMSCs)in patients with steroid-induced osteonecrosis of femoral head(SONFH),and to explore the correlation between the expression of H19 and DNMTs.Methods: Patients with SONFH preparing for total hip arthroplasty(THA)were selected as ONFH Group,and patients with femoral neck fractures(FNF)were selected as Control Group,and BMSCs from the two groups were isolated,cultured and identified.Firstly,lnc RNA sequencing analysis was used to screen the differentially expressed lnc RNAs in undifferentiated BMSCs of the two groups.Subsequently,the expression patterns of H19 and DNMTs in undifferentiated and osteogenic/adipogenic-differentiated BMSCs in the Control Group,ONFH Group and DNMTs inhibitor 5-aza-d C treatment group were detected by q RT-PCR,and the methylation status of the H19 promoter region was analyzed by bisulfite-sequencing PCR(BSP)assay.Immunofluorescence(IF)staining and PCR analyses of fractionated nuclear and cytoplasmic RNA were used to determine the functional subcellular localization of Dnmt1 and H19.Results: BMSCs in both groups highly expressed the cell surface markers,including CD73,CD90 and CD105,and lowly expressed CD34 and HLA-DR.These cells had the trilineage differentiation ability of osteogenic,adipogenic and chondrogenic differentiation.Lnc RNA sequencing analysis showed that the expression of H19 in BMSCs of ONFH Group was significantly higher than that in Control Group.BSP assay and q RT-PCR revealed that hypomethylation of H19 promoter along with significantly increased H19 expression and downregulated Dnmt1 expression were observed in undifferentiatedBMSCs of ONFH Group,while significantly upregulated Dnmt1 expression and downregulated H19 expression were observed in these cells differentiated into osteogenic and adipogenic lineage,and the cells of ONFH Group treated with 5-aza-d C resulted in significantly elevated H19 expression.In addition,IF staining indicated that Dnmt1 was predominantly located in the nuclear in undifferentiated BMSCs of both Control Group and ONFH Group,while lower fluorescence intensity of Dnmt1 was observed in cells of ONFH Group than in cells of Control Group.PCR analyses of fractionated nuclear and cytoplasmic RNA revealed that H19 was localized in both the nuclear and cytoplasmic compartment in undifferentiated BMSCs of Control Group and ONFH Group,while H19 was mainly distributed in the nucleus in cells of ONFH Group and predominantly localized in the cytoplasm in cells of Control Group.Conclusion: Therefore,the expression patterns of DNMTs and H19 in BMSCs indicated that the expression of H19 and Dnmt1 in either undifferentiated or osteogenic/adipogenic-differentiated BMSCs between Control Group and ONFH Group were negatively correlated,while the expression patterns of H19 was not significantly correlated with that of Dnmt3 a and Dnmt3 b.The administration of glucocorticoids may be an important trigger for the transport of H19 from cytoplasm to the nucleus in undifferentiated BMSCs.Part Ⅱ: Roles of DNMTs in regulating H19 expression as well as osteoblastogenesis and adipogenesis of BMSCs from SONFH patientsObjective: By knocking down DNMTs or overexpressing Dnmt1 in BMSCs from patients with SONFH,the roles of DNMTs in regulating H19 expression as well as osteoblastogenesis and adipogenesis of BMSCs were explored.Methods: Firstly,si RNA transfection and plasmid transfection were used to knockdown the expression of DNMTs and overexpress the expression of Dnmt1 in BMSCs of ONFH Group,respectively.Successful knockdown of DNMTs and overexpression of Dnmt1 were verified by Western blot analysis,and then the altered expression of H19 and methylation status of the H19 promoter region after knockdown of DNMTs or overexpression of Dnmt1 in BMSCs of ONFH Group were detected by q RT-PCR and BSP analysis,respectively.Secondly,BMSCs of ONFH Group were treated with 5-aza-d C.Following 5-aza-d C treatment,the alterations in the expression of the osteogenic markers RUNX2 and COL1A1 during osteogenic differentiation and the adipogenic markers FABP4 and PPARγ during adipogenic differentiation were detected by q RT-PCR,and then the alterations in mineralized nodule formation and intracellular lipid accumulation were detected by Alizarin Red S(ARS)and Oil Red O(ORO)staining and quantification.In addition,osteogenic and adipogenic induction experiments of BMSCs were performed after knocking down DNMTs or overexpressing Dnmt1 in BMSCs of ONFH Group.Subsequently,the alterations in mineralized nodule formation and intracellular lipid accumulation were evaluated using ARS and ORO staining and quantification,respectively,and the alterations in the protein expression levels of the osteogenic markers and adipogenic markers were detected by Western blot analysis.Results: Firstly,Dnmt1 knockdown in BMSCs of ONFH Group led to hypomethylation of the H19 promoter region and significantly increased the expression of H19,and overexpression of Dnmt1 resulted in hypermethylation of the H19 promoter region and significantly reduced the expression of H19,while Dnmt3 a or Dnmt3 b knockdown had no significant effect on the expression of H19.Secondly,the results of q RT-PCR indicated that 5-aza-d C treatment significantly increased the expression levels of osteogenic markers RUNX2 and COL1A1 after 3,7 and 14 days of osteogenic induction,and significantly decreased the expression levels of adipogenic markers FABP4 and PPARγ at days 3,7 and14 after adipogenic induction.Furthermore,ARS and ORO staining and quantification showed that 5-aza-d C treatment significantly increased mineralized nodule formation,and significantly reduced intracellular lipid accumulation.Additionally,Dnmt1 knockdown in BMSCs of ONFH Group,but not Dnmt3 a or Dnmt3 b knockdown,significantly increased the number of mineralized nodules and the protein expression levels of osteogenic markers,and significantly reduced intracellular lipid accumulation and the protein expression levels of adipogenic markers,while overexpression of Dnmt1 showed the opposite effects.Conclusion: The three DNMTs have different roles in regulating H19 expression as well as osteoblastogenesis and adipogenesis of BMSCs from SONFH patients,that is,Dnmt1 is the key DNMTs regulating H19 expression at the transcriptional levels as well as osteoblastogenesis and adipogenesis of BMSCs,while Dnmt3 a and Dnmt3 b have no significant regulatory effect.Part Ⅲ: Roles of H19 in regulating osteogenesis and adipogenesis as well as Wnt/β-catenin signaling pathway of BMSCs from SONFH patientsObjective: By knocking down and overexpressing H19 in BMSCs from patients with SONFH,the roles of H19 in regulating osteoblastogenesis and adipogenesis as well as Wnt/β-catenin signaling pathway were explored.Methods: Firstly,si RNA transfection and plasmid transfection were used to knockdown and overexpress the expression of H19 in BMSCs of ONFH Group,respectively,and the efficiency of knockdown or overexpression was verified by q RT‐PCR.Then,BMSCs with knocked down or overexpressed H19 were subjected to osteogenic or adipogenic induction differentiation.Subsequently,the alterations in mineralized nodule formation and intracellular lipid accumulation were evaluated using ARS and ORO staining and quantification,respectively,and the alterations in the protein expression levels of the osteogenic markers and adipogenic markers were detected by Western blot.Furthermore,the alterations in the protein expression of p-Ser9 GSK-3β/Total GSK-3β and Activeβ-catenin/Totalβ-catenin in BMSCs with knocked down or overexpressed H19 were detected by Western blot.BMSCs transfected with H19 si RNA(si H19 group)or its negative control(si CTL group)were treated with GSK-3β inhibitor Tideglusib,and then osteogenic or adipogenic induction differentiation was performed.Following treatment with Tideglusib,the alterations in mineralized nodule formation and intracellular lipid accumulation were evaluated using ARS and ORO staining and quantification,respectively,and the alterations in the protein expression of Active β-catenin/Totalβ-catenin and osteogenic markers and adipogenic markers were detected by Western blot.Results: Firstly,ARS and ORO staining and quantification showed that H19 knockdown in BMSCs of ONFH Group led to a significant decrease in the mineralized nodule formation and a significant increase in the intracellular lipid accumulation,while overexpression of H19 showed the opposite effects.Furthermore,the results of Western blot analysis indicated that H19 knockdown led to a marked decrease in the protein expression of osteogenic markers and a significant increase in the protein expression of adipogenic markers,while the effects of H19 overexpression were the opposite to H19 knockdown.Secondly,H19 knockdown in BMSCs of ONFH Group led to a significant decrease in the relative protein expression of p-Ser9 GSK-3β/Total GSK-3β and Activeβ-catenin/Total β-catenin,while overexpression of H19 led to a significant increase in the relative protein expression of them.Thirdly,compared to the corresponding group without Tideglusib treatment,treatment of BMSCs of si CTL group or si H19 group with Tideglusib led to a significant increase in the protein expression of Active β-catenin/Total β-catenin and osteogenic markers,and a significant decrease in adipogenic markers.Additionally,the protein expression of Active β-catenin/Total β-catenin and osteogenic markers in the si H19 group treated with Tideglusib was significantly lower than that in the si CTL group treated with Tideglusib,while the protein expression of adipogenic markers in the si H19 group treated with Tideglusib was significantly higher than that in the si CTL group treated with Tideglusib.Conclusion: H19 inhibits the activity of GSK-3β by promoting the phosphorylation of GSK-3β at Ser9 site,and the inactivation of GSK-3β leads to suppression of β-catenin degradation in cytoplasm and subsequent an increase in β-catenin entering the nucleus,which activates the Wnt/β-catenin signaling pathway,resulting in the enhanced osteogenesis and weakened adipogenesis of BMSCs from SONFH patients.Part Ⅳ: The therapeutic effects of intramedullary injection with BMSCs of Dnmt1 knockdown or H19 overexpression on early-stage SONFH in ratsObjective: By knocking down Dnmt1 or overexpressing H19 in BMSCs of early-stage SONFH in rats,the therapeutic effects of intramedullary femur injection with r BMSCs of Dnmt1 knockdown or H19 overexpression on early-stage SONFH in rats were explored.Methods: Firstly,SONFH model in rats was established by the administration of methylprednisolone(MPS)in combination with lipopolysaccharide(LPS),and BMSCs were isolated and cultured from MPS-treated rats(MPS group)and age-matched normal control rats(NC group).Secondly,lentiviral transfection was performed to knockdown Dnmt1(sh-Dnmt1)or overexpress H19(oe-H19),and the effectiveness of Dnmt1 silencing and H19 overexpression was verified by Western blot and q RT-PCR,respectively.Subsequently,according to different treatments via intramedullary femur injection,the rats were assigned to five groups(n=6 per group): NC+Vehicle(PBS injection)group,MPS+Vehicle group,MPS+Lenti-Ctrl(sh-Ctrl or oe-Ctrl)BMSCs group,MPS+sh-Dnmt1 or oe-H19 BMSCs group,MPS+NC BMSCs group.The intra-femoral injection of rats in different groups was carried out 4 weeks and 6 weeks after the induction of the ONFH model,respectively.At 6 weeks after the intramedullary femur injection,the immunohistochemical(IHC)staining of Brd U,an exogenous cell tracer,was applied to detect the distribution of Brd U-labeled rat BMSCs within the femoral head.Subsequently,Micro-CT,hematoxy-eosin(H&E)staining,along with IHC staining for osteogenic and adipogenic markers were performed to evaluate the therapeutic effects of intramedullary femur injection with BMSCs of Dnmt1 knockdown or H19 overexpression on early-stage SONFH in rats.Results: Firstly,abnormally high signal intensity on the T2-weighted image,significant destruction of trabecular structure and subchondral cystic degeneration,and diffused empty lacunae or pyknotic nucleus of osteocytes in trabecular bone were observed in the femoral head of MPS group rats,as indicated by Micro-MRI,Micro-CT and H&E staining,respectively.These results confirmed the successful establishment of the SONFH rat model.Secondly,the results of Western blot showed that lentiviral transfection significantly reduced the expression of Dnmt1 in rat BMSCs of the MPS group.The results of q RT-PCR indicated that lentiviral transfection significantly increased the expression of H19 in rat BMSCs of the MPS group.Thirdly,IHC staining showed that 6weeks after rat BMSCs transplantation,Brd U-positive cells were widely distributed in the cartilage,subchondral region,bone marrow cavity,and trabecular bone of the femoral head.The results of Micro-CT showed that MPS-treated rats either implanted with sh-Dnmt1 or oe-H19 r BMSCs significantly increased trabecular number(Tb.N),bone volume per tissue volume(BV/TV)and trabecular thickness(Tb.Th),and markedly decreased trabecular separation(Tb.Sp),that is,markedly improved the destruction of trabecular microstructure in the femoral head.H&E staining indicated that MPS-treated rats either implanted with sh-Dnmt1 or oe-H19 r BMSCs significantly reduced the numbers of empty lacunae and pyknotic nucleus of osteocytes.IHC staining revealed that the expression of osteogenic markers COL1A1 and OCN were markedly increased while the expression of adipogenic markers FABP4 and PPARγ were significantly decreased in the femoral head of MPS-treated rats either implanted with sh-Dnmt1 or oe-H19 rBMSCs.Conclusion: Transplantation of genetically modified Dnmt1 knockdown or H19over-expressing BMSCs by intramedullary femur injection in SONFH rat model promotes bone regeneration and reduces fat accumulation in the necrotic region of the femoral head,thereby effectively delays even blocks the disease progression. |
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