Mechanism Of YTHDF1 Promoting Osteogenic Differentiation Of MC3T3-E1 Cells Through Post-Transcriptional Regulation Of THBS1 Under Hypoxia

Objective:Peri-implantitis（PI）is a pathological state of peri-implant tissues caused by bacterial plaque,manifested by peri-implant mucosal inflammation and persistent destruction of supporting bone tissue,bleeding and pus spillage on probing,increased probing depth and bone loss on radiographic imaging,and is the main cause of implant loosening failure,while the specific anatomy of the peri-implant area makes it more The specific anatomy around the implant makes it more susceptible to the local hypoxic environment caused by inflammation.Peri-implant tissues exhibit a higher risk of inflammation because they lack two important components of periodontal tissue:the bound epithelium and periodontium as well as the cytoarchitecture and vascular structures[1].Due to the lack of periodontal membrane around the implant and the reduced blood supply,the host response and immune cell migration in peri-implant tissues are more restricted compared to natural teeth.Many of the etiologies and pathogenesis of peri-implant disease are currently considered similar to periodontitis.However,unlike periodontitis,peri-implant lesions show a poorer vascular supply and a more extensive distribution of inflammatory cells such as B cells,osteoblasts and neutrophils.Thus peri-implantitis shows a greater susceptibility to inflammation-induced hypoxia,and it is clinically important to study the pathogenesis of peri-implantitis under hypoxia.N6-methyladenosine（m6A）RNA methylation is an emerging molecular mechanism that plays an important role in numerous biological processes and can regulate gene expression at the post-transcriptional level.Typically this modification process is a dynamic and reversible one,regulated by three m6A key factors.These are methylation transferases and demethylases,which deposit or remove methyl residues from the N6atom of m RNA adenosine,and RNA binding proteins（RBPs）,which interact with gene transcripts to regulate RNA maturation,transport,stability and degradation.m6A readers can regulate a variety of biological processes to recognise and bind to m6A-modified m6A readers can regulate gene expression by regulating a variety of biological processes to recognise and bind to m6A-modified transcripts,m RNA is dynamically and reversibly modified by m6A by methyltransferases and demethyltransferases,and the’readers’can recognise and function with methylated RNA.In addition,several different’readers’of m6A can specifically recognise m6A modifications and fulfil many biological functions.Among them,YTH m6A RNA binding protein 1（YTHDF1）plays an important role in many biological processes by enhancing m RNA stability and interacting with translation initiation factors and ribosomes to facilitate translation.It has been reported that YTHDF1 is elevated in hypoxic environments such as breast and liver cancer and can promote osteogenic differentiation of human bone marrow mesenchymal stem cells.However,the effect of YTHDF1 on osteogenic differentiation under hypoxic conditions remains to be investigated.Thrombospondin-1（THBS1）,a glycoprotein whose expression level is significantly increased in hypoxia and whose m RNA is predicted to be a downstream site for YTHDF1 methylation recognition,promotes osteogenesis in MC3T3-E1 cells under both normoxia and hypoxia.In conclusion,the present study was designed to investigate the mechanism of osteogenic differentiation regulation by MC3T3-E1 cells in the hypoxic environment generated by peri-implantitis from the perspective of post-transcriptional RNA modification,and to explore new therapeutic targets for hypoxia-induced osteogenic inhibition in peri-implantitis.Methods:1.The differential expression of YTHDF1 was examined using the microarray data retrieved from the GEO database,and MC3T3-E cells were treated at three different oxygen concentrations（21%,5%,1%）to simulate the hypoxic environment during the development of peri-implantitis,and the expression of hypoxia-inducible factor-1α（HIF-1α）and YTHDF1 at different oxygen concentrations was examined to measure the cell The expression of hypoxia-inducible factor-1α（HIF-1α）and YTHDF1 was measured at different oxygen concentrations to measure the degree of hypoxia and determine the most suitable hypoxic concentration.The expression of ALP,RUNX2and COL1,markers of osteoblast differentiation,alkaline phosphatase activity（ALP）and alizarin red staining（ARS）were observed by RT-q PCR and Western blot for osteogenic activity and extracellular matrix mineralization（EMM）nodules under normoxia and hypoxia.2,YTHDF1 was knocked down in normoxia and hypoxia,and osteogenesis-related indexes and alkaline phosphatase activity（ALP）and alizarin red staining（ARS）levels were observed by RT-q PCR method and Western blot.Bioinformatics analysis was performed to predict the factors associated with YTHDF1 binding through m6A modification,and actinomycin D assay was performed to detect THBS1 m RNA stability under normoxia and hypoxia.Immunofluorescence（IF）was used to detect the co-localization of YTHDF1 with THBS1 under normoxia and hypoxia.3,Knockdown of THBS1 under normoxia and hypoxia,osteogenesis-related indexes and alkaline phosphatase activity（ALP）and alizarin red staining（ARS）levels were observed by RT-q PCR and Western blot.Osteogenic activity was observed by Western blot and alkaline phosphatase activity（ALP）under normoxia and hypoxia.Results:1,GEO database results showed increased expression of YTHDF1 in peri-implantitis compared to normal tissue,with the highest expression of HIF-1αat 1%oxygen concentration and peak expression of YTHDF1 in hypoxic culture for 24 h.Therefore,1%O₂ incubation for 24 h was determined as a depleted oxygen condition.The m RNA expression of marker genes related to osteogenic differentiation,including Alpl,Runx2,and Col1a1,decreased under hypoxic conditions（p<0.05）,and the protein levels of key transcription factors Alp,Runx2,and collagen type 1（Col1）were also confirmed to be inhibited by hypoxia（p<0.05）.Alkaline phosphatase activity（ALP）and alizarin red staining（ARS）also showed that hypoxia decreased the osteogenic activity of MC3T3-E1 cells.2,RT-q PCR and Western blot results showed that knockdown of YTHDF1 decreased the expression of osteogenic-related genes.Based on the results obtained from bioinformatics and data from GO database,GO biological process（BP）analysis of YTHDF1 and osteogenic target genes showed that THBS1 was enriched to the genes associated with hypoxic response and was the most increased m6A modification recognized by YTHDF1.m RNA stability assay showed that silencing YTHDF1significantly decreased THBS1 m RNA stability was significantly reduced under both normoxic and hypoxic conditions.Immunofluorescence co-localization staining experiments showed that YTHDF1 co-localized with THBS1 in the cytoplasm under hypoxia.3,Loss-of-function studies showed that knockdown of YTHDF1 or THBS1exacerbated hypoxia-induced osteogenic inhibition.Overexpression of YTHDF1upregulated osteogenesis-related Under normoxic and hypoxic conditions,silencing THBS1 reversed the upregulation of osteogenesis-related protein expression of Alp,Runx2 and Col1 caused by YTHDF1 overexpression.Conclusion:1,Hypoxia inhibited the osteogenic differentiation of MC3T3-E1 cells and promoted the expression of YTHDF1 and THBS1.2,YTHDF1,THBS1 may promote osteogenic differentiation of MC3T3-E1 cells under normoxic or hypoxic conditions,respectively or via YTHDF1/THBS1.3,Methylated THBS1 m RNA can be recognized and combined by YTHDF1,thereby enhancing the stability of THBS1 m RNA and promoting translation,resulting in elevated THBS1 expression levels that act as a downstream factor of YTHDF1 to co-resist the downregulation of osteogenic differentiation under hypoxia,providing a promising therapeutic strategy for bone resorption in hypoxia-induced peri-implantitis.