The Molecular Mechanism Of Lnc-ODPR1 Promoting The Osteogenesis Of MSCs

PurposeDefects or dysfunction of tissue and organ caused by trauma,disease,aging or genetics in China rank first in the world.Among of it,more than3 million patients every year suffer from defects or dysfunction of bone caused by trauma,fractures,and spinal degenerative diseases and so on.Therefore,it is urgent to explore new strategy for bone regeneration and repair."Stem cell therapy",which focuses on stem cells with self-renewal and multidirectional differentiation potential,is a promising new strategy in the regenerative medicine field.Stem cell therapy repairs the damaged site through stem cells transplanted to the damaged site themselves differentiating or inducing progenitor/precursor cells to differentiate into new functional cells in vivo.The essence of bone regeneration and repair is the directional differentiation of stem cells into osteoblasts,so it is crucial to identify the key regulatory factors in the osteogenesis process of stem cells.We discovered and identified a novel lnc RNA,osteogenic differentiation promoting lnc RNA 1(Lnc-ODPR1)through high throughput sequencing and RT-q PCR.We found that Lnc-ODPR1 was upregulated during osteogenic differentiation of mesenchymal stem cells(MSCs),and overexpression of Lnc-ODPR1 promoted osteogenic differentiation in vitro and bone repair after defect in vivo.Therefore,we will delve into the role and molecular mechanism of Lnc-ODPR1 in osteogenic differentiation of MSCs in this study,providing new targets and experimental data for the bone regeneration and repair strategy.MethodsBy detecting the molecular expression of umbilical cord mesenchymal stem cells and dental pulp mesenchymal stem cells after osteogenesis induction,it was confirmed that the expression level of Lnc-ODPR1 was upregulated in the osteogenic differentiation of MSCs.The cell lines with stable overexpression and stable knockdown of Lnc-ODPR1 were constructed respectively,and the osteogenic differentiation efficiency was detected by alkaline phosphatase staining and alizarin red calcium nodule staining after osteogeneis of cell lines.The intracellular distribution of Lnc-ODPR1 was analyzed by RNA nucleocytoplasmic separation-RT-q PCR and fluorescence in situ hybridization assays.The specific binding protein of Lnc-ODPR1 was identified by RNA pull down and mass spectrometry,and then it was reversely verified by RNA immunoprecipitation assay.The interaction between proteins was confirmed by protein immunoprecipitation experiment.RT-q PCR and Western blot assays were used to detect the m RNA and protein levels of molecules.Finally,the ability of Lnc-ODPR1 to promote osteogenesis and bone repair after defect in vivo was confirmed by the experiment of repairing femoral defect in nude mice.Results1.The expression level of Lnc-ODPR1 was upregulated in the osteogenic differentiation of UC-MSCs and DP-MSCs;2.Overexpression of Lnc-ODPR1 promoted the expression of the key osteogenic factor OSX and enhanced the alkaline phosphatase(ALP)staining level and alizarin red calcium nodule(ARS)staining level after osteogenic differentiation,and knockdown of Lnc-ODPR1 inhibited the expression of OSX and reduced the ALP staining level and ARS staining level after osteogenic differentiation;knockdown of Lnc-ODPR1 downregulated expression level of Distal less homeobox 5(Dlx5);3.Lnc-ODPR1 interacted with poly(ADP-Ribose)glycohydrolase(PARG)and valosin-containing protein(VCP);Lnc-ODPR1 stabilized PARG protein by inhibiting its proteasome degradation,without affecting its m RNA level;knockdown of PARG inhibited the expression of OSX and reduced the level of ALP staining and ARS staining after osteogenic differentiation;PARG protein interacted with Smad1/5 protein and overexpression of Lnc-ODPR1 reduced ADP-ribosylation of Smad5;4.Lnc-ODPR1 had no influence on m RNA and protein of VCP;VCP stabilized PARG protein through binding to PARG protein and inhibiting its proteasome degradation;and overexpression of Lnc-ODPR1 promoted the interaction between VCP and PARG proteins,knockdown of Lnc-ODPR1 inhibited the interaction between VCP and PARG proteins,and RNase treatment eliminated the interaction between VCP and PARG proteins;5.Lnc-ODPR1 positively regulated the expression of m RNA and protein of N-6 adenine-specific DNA methyltransferase 1(N6AMT1),and N6AMT1 positively regulated the expression of Lnc-ODPR1;N6AMT1 did not affect the m RNA and protein levels of VCP;N6AMT1stabilized PARG protein by inhibiting its proteasome degradation;and knockdown N6AMT1 inhibited the expression of OSX and reduced the levels of ALP staining and ARS staining after osteogenic differentiation;6.Both Lnc-ODPR1 and N6AMT1 upregulated m RNA and protein levels of BDNF and promoted phosphorylation of ERK1/2 and AKT;7.The bone repair assay after defect in nude mice confirmed that Lnc-ODPR1 increased expression of N6AMT1 and PARG,and promoted the bone repair after defect.Conclusions1.Lnc-ODPR1 was highly expressed in the osteogenic differentiation of MSCs,and promoted the osteogenic differentiation of MSCs by up-regulating the key osteogenic factor OSX;2.Lnc-ODPR1 promoted interaction of VCP and PARG as a scaffold which resulted in inhibited proteasome degradation of PARG protein,stabilized PARG protein and de ADP-ribosylation of Smad1/5,and then activated Dlx5 transcription,thereby promoting OSX expression;3.Lnc-ODPR1 regulated N6AMT1 with positive feedback,and N6AMT1 participated in Lnc-ODPR1 mediating osteogenic differentiation through PARG.Lnc-ODPR1 also upregulated BDNF and promoted phosphorylation of ERK1/2 and AKT through N6AMT1,thereby promoting OSX expression.