A Peptide RIP Encoded By LncRNA DGCR5 Regulates Differentiation Lineage Of Mesenchymal Stem Cell Leading To The Progression Of Steriod-Induced Osteonecrosis Of The Femoral Head

Backgound:Osteonecrosis of the femoral head(ONFH)is a serious orthopedic refractory disease.Unknown pathogenesis and lack of specific therapeutic targets are the main reasons for the refractory disease.Steroid-induced osteonecrosis of the femoral head(SONFH)accounts for 40%of the overall incidence and is the most important pathogenic factor of ONFH.A large number of adipocytes can be accumulated in the necrotic bone tissue of SONFH patients,and bone marrow-derived mesenchymal stem cells(mesenchymal stem cells,MSCs)in SONFH patients have abnormally enhanced adipogenic differentiation.Studies have found that in animal models of SONFH,translations of MSCs induced by adipogenic differentiation into the bone marrow cavity can effectively promote the progression of SONFH.Therefore,enhanced adipogenic differentiation of MSCs is considered to be an important reason for the progression of SONFH.Clinical studies have also shown that the abnormally enhanced adipogenic differentiation of bone marrow-derived MSCs may be the reason for the poor curative effect of patients with early SONFH after cardiac decompression combined with autologous bone marrow MSC transplantation.Steriod-induced regulation in epigenetic mechanisms are considered to be the main pathogenesis of SONFH.Long non-coding RNAs(long non-coding RNAs,lncRNAs)are of great significance in regulating the direction of MSC differentiation at the epigenetic level,and targeted therapeutic drugs designed for lncRNAs also have good clinical application prospects.However,in recent years,miRNA treatment programs designed based on the classic mechanism of lncRNAs have had little effect in the treatment of SONFH animal models.Recently,a large number of authoritative journals have reported that in plant and animal cells,some lncRNAs can play a role in the regulation of epigenetic mechanisms by encoding functional polypeptides.In MSCs,lncRNAs may regulate the direction of their differentiation lineage through similar non-canonical mechanisms.In-depth analysis of the exact mechanism of lncRNAs regulating MSC differentiation lineage at the epigenetic level will help to find new targets for clinical treatment of SONFH.In this study,through bioinformatics analysis and experimental verification of MSC sequencing data,it was found that the highly expressed LncRNA DGCR5 in SONFH MSCs can promote their adipogenic differentiation.Then,the molecular structure was analyzed and the potential open reading frame(open read frame,ORF)was predicted.It was confirmed through experiments that LncRNA DGCR5 affected the direction of MSC differentiation lineage by encoding the functional polypeptide RIP(RAC1 inactive peptide,RIP).Then,Co-Immunoprecipitation(Co-IP)combined with mass spectrometry identification technology was used to identify RIP downstream interacting proteins,explore the specific mechanism of RIP promoting MSC adipogenic differentiation,and provide new targets for the treatment of SONFH.Method:By analyzing the MSC transcriptome sequencing data combined with the verification of SONFH clinical samples,LncRNA DGCR5 was found significantly over-expressed in MSC which promoting adipogenic differentiation.further analysis of its molecular structure,subcellular localization and potential open reading frame was used to analyze the coding of LncRNA DGCR5 Possibility of functional polypeptides,then by constructing a specific plasmid vector to transfect HEK293T cell line,explore the translation activity of the initiation codon ATG of the potential open reading frame.MSCs were transfected with specific overexpression plasmid vectors and knockdown vectors,and then cultured for osteogenic and adipogenic differentiation respectively.Through qPCR,WB,Alizarin Red S staining and Oil Red O staining to identify the role of the functional polypeptide encoded by the open reading frame on the MSC differentiation lineage,and to identify the effect of knocking down LncRNA DGCR5 on the MSC differentiation lineage.Co-immunoprecipitation(Co-Immunoprecipitatio,Co-IP)combined with mass spectrometry identification technology was used to find downstream interacting proteins of RIP.through sequencing analysis of MSC transcriptomes overexpressing RIP,it was found that the Wnt/β-catenin signaling pathway changed significantly.Co-IP combined with mass spectrometry identified the downstream interaction protein RAC1.Co-IP was used to verify the interaction between RIP and RAC1.The RAC1 truncated mutant plasmid vectors were constructed to further clarify the binding region of the two.The activation level of RAC1 andβ-catenin Ser675 phosphorylation level was then detected.Meanwhile,subcellular localization of β-catenin was detected to clarify the role of RIP on P-catenin stability and subcellular localization.Finally,by detecting the activation level of TCF/LEF1,the activation of Wnt/β-catenin signaling pathways was clarified.By designing the rescue experiment,the rescue effect of RAC 1 in MSC and SONFH models of rats was verified.Results:1.A large number of adipocytes accumulate in the femoral head necrosis area of SONFH,and LncRNA DGCR5 is upregulated in SONFH bone marrow-derived MSCs,which promotes their adipogenic differentiation;2.ORF2 of LncRNA DGCR5 has the ability to encode polypeptides,its start codon ATG has translation activity,and the expression of polypeptide RIP is up-regulated in bone tissue of SONFH patients;3.LncRNA DGCR5 regulates MSC adipogenesis enhancement and osteogenesis inhibition by encoding polypeptide RIP,and promotes the disease progression in SONFH rats;4.RIP binds to the N-terminal motif of RAC1,down-regulates its activation level and downstream P-catenin Ser675 phosphorylation level and subcellular localization,and finally inhibits the transcriptional activation level of TCF/LEF1;5.Overexpression of RAC1 can antagonize the role of RIP in promoting MSC adipogenicity and inhibiting osteogenic differentiation,and delay the progression of the disease in SONFH rats.Conclusion:1.The expression of LncRNA DGCR5 was significantly upregulated in MSCs derived from SONFH patients;2.LncRNA DGCR5 regulates MSC adipogenesis enhancement and osteogenesis inhibition by encoding polypeptide RIP;3.RIP down-regulates the phosphorylation level of β-catenin Ser675 by inhibiting the activation of RAC1,thereby reducing the nuclear localization of β-catenin;4.Overexpression of RAC1 can antagonize the effect of RIP on promoting MSC adipogenesis and inhibiting osteogenesis.