| IntroductionBone development and bone repair depend on the differentiation of mesenchymal stem cells into osteoblast progenitor cells-osteogenic precursor cells-osteocyte lineages.,Osterix;Cytokines BMP,Wnt,FGF;Bone matrix protein synthesis Col ?,OCN,BSP.More and more researchers are paying attention,and its transformation to bone development and bone repair is gradually being imitated.Long non-coding RNA(LncRNA)is a type of RNA that exceeds 200 nt and has almost no coding ability.At first,LncRNA was regarded as the "noise" of genome transcription.It was a by-product of RNA polymerase ? transcription and did not have biological functions.However,it has been confirmed that LncRNA is widely involved in chromosome silencing,genome imprinting,chromatin modification,and transcription activation.Transcription interference.Recently,LncRNA in bone development and bone repair has gradually attracted attention.The main research and exploration of this study is the osteogenic differentiation process of LncRNA HOTTIP in bone marrow mesenchymal stem cells discovered through DNA microarray(microarray)analysis.The role and molecular mechanism in the process.Objective:To identify the effect of LncRNA HOTTIP on osteogenic differentiation by overexpression and knockdown of LncRNA HOTTIP in bone marrow mesenchymal stem cells,and explore the downstream signaling pathways through which LncRNA HOTTIP promotes osteogenic differentiation in the process of osteogenic differentiation,and explore how LncRNA HOTTIP promotes the osteogenic differentiation of bone marrow mesenchymal stem cells in vivo.Methods:(1)Use Ficoll lymphocyte separation fluid to extract human bone marrow mesenchymal stem cells(BMSC),and culture and expand.Use morphological observation and flow cytometry to detect cell surface markers to identify the purity of the cells;(2)To induce osteogenic differentiation of the cells,extract RNA from the kit,and perform gene chip detection to obtain data after quality control,and to dig deeper into the data Analyze the highly expressed LncRNA;(3)Use PCR and Western Blot technology to detect the expression of HOTTIP and the expression of the osteogenic marker Runx2 during the osteogenic differentiation of BMSCs,and use Alizarin Red staining to determine the final cell mineralization,Use shRNA to knock down HOTTIP in BMSCs,use PCR technology to verify the success of knockdown,use Alizarin Red staining and quantitatively determine the mineralization changes of BMSCs after knocking down HOTTIP,and use WB technology to detect the results.Changes in bone indicators ALP,Runx2 and Osterix;(4)Overexpression of HOTTIP in BMSCs using overexpression lentivirus,PCR technology to verify the success of overexpression,Alizarin red staining and quantitative judgment of BMSCs after HOTTIP overexpression WB technology is used to detect changes in osteogenic indicators ALP,Runx2,and Osterix.(5)After HOTTIP was overexpressed in bone marrow mesenchymal stem cells(BMSC)by overexpression lentivirus,the changes of downstream ?-catenin were detected by PCR,Western Blot,and immunofluorescence technology;Construct BMSCs overexpressing HOTTIP stably,use shRNA to knock down ?-catenin and use XAV(?-catenin inhibitor)to inhibit the activity of ?-catenin in BMSCs overexpressing HOTTIP,and use alizarin red staining and quantitative detection of BMSCs Mineralization,the changes of osteogenic indicators were detected by PCR and WB.Use shRNA to knock down WDR5 in bone marrow mesenchymal stem cells(BMSC)overexpressing HOTTIP stably,verify the knockdown efficiency by PCR technology,induce osteogenic differentiation of BMSC overexpressing HOTTIP,and stain with Alizarin Red And quantitatively detect the mineralization of BMSC,and use PCR and Western Blot technology to detect the changes of osteogenic indicators;Use overexpression lentivirus to stably knock down HOTTIP bone marrow mesenchymal stem cells(BMSC)Overexpression of WDR5,the efficiency of overexpression was verified by PCR technology,the osteogenic differentiation of BMSCs knocked down HOTTIP was induced,and the mineralization of BMSCs was detected through Alizarin Red staining and quantification;In the bone marrow that knocked down WDR5 Using PCR technology to detect the mRNA expression of ?-catenin in mesenchymal stem cells(BMSC);Using fluorescence in situ hybridization(FISH)to detect cell sublocalization of HOTTIP;Using immunofluorescence technology to detect WDR5 Cell sublocalization detection.Use RNA immunoprecipitation and RNA pull down techniques to detect the binding of HOTTIP and WDR5;(6)Use the ChIP experiment to detect the binding of WDR5 to the ?-catenin promoter.(7)The bone marrow mesenchymal stem cells(BMSC)stably overexpressing HOTTIP were constructed,the BMSCs were cultured on collagen scaffolds in vitro,and the collagen scaffolds were transplanted under the skin of nude mice,and XAV(?-catenin inhibitor)was given to interfere with ?-catenin The activity of catenin is finally detected by ?CT for the size and density of bone formation,and the expression of the osteogenic index OCN is detected by immunohistochemistry(IHC)technology.Results:(1)We found that HOTTIP increased in the differentially expressed LncRNA microarray map;HOTTIP was gradually up-regulated during the osteogenic differentiation of BMSCs;HOTTIP knocked out inhibited the osteogenic differentiation of human bone marrow mesenchymal stem cells;overexpression of HOTTIP promoted Osteogenic differentiation of human bone marrow mesenchymal stem cells.(2)LncRNA HOTTIP activates the Wnt/?-catenin pathway in the process of osteogenic differentiation,and promotes the increase of ?-catenin expression and nuclear localization;knocking down ?-catenin and inhibiting ?-catenin activity will cause HOTTIP to promote osteogenic differentiation.The effect is reduced.Knockdown of WDR5 will result in the reduction of the osteogenic differentiation effect of HOTTIP,while overexpression of WDR5 can save the reduction of the osteogenic differentiation effect of the knockdown of HOTTIP;WDR5 can promote the transcription of ?-catenin;in the process of BMSC osteogenic differentiation,The nuclear localization of HOTTIP increases;HOTTIP and ?-catenin bind to each other;after knocking down HOTTIP,the nuclear localization of WDR5 decreases,and its binding to ?-catenin promoter also decreases.(3)Compared with the control group,BMSCs stably overexpressing HOTTIP had larger bone formation volume,higher bone density,and more expression of osteogenic index OCN after transplantation into mice;while XAV was given to interfere with ?-catenin activity The bone formation effect brought by HOTTIP was reduced,and the bone formation volume,bone density,and osteogenic index OCN expression were not as good as those in the HOTTIP overexpression group.Conclusion:LncRNA HOTTIP plays an important role in the osteogenic differentiation of BMSCs.HOTTIP promotes the osteogenic differentiation of human bone marrow mesenchymal stem cells.LncRNA HOTTIP participates in the activation of Wnt/?-catenin,and its effect on promoting BMSC differentiation depends on the effect of ?-catenin.LncRNA HOTTIP promotes the osteogenic differentiation of bone marrow mesenchymal stem cells in vivo and depends on the activation of the Wnt/?-catenin pathway. |
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