Effect Of Mir-34a-5p On The Osteogenic Differentiation Of RBMSCs In Inflammatory Microenvironment And Related Mechanism

Background and ObjectivePeriodontitis is a common chronic inflammatory disease of the oral cavity,which leads to the loss of teeth due to the progressive destruction of periodontal support tissue.Bone marrow mesenchymal stem cells(BMSCs)are a kind of mesenchymal stem cells with strong self-renewal and multidirectional differentiation potential.Stem cells have the potential of bone differentiation,which is an important cytological basis of bone formation and makes periodontal tissue regeneration possible.However,the continuous effect of inflammation inhibits the differentiation of mesenchymal stem cells into bone and affects the formation of new bone.Bone defects are one of the global health problems and the current treatment is still facing great challenges.In addition to the common clinical bone defects caused by trauma,a series of bone diseases caused by age,infection,systemic diseases,such as bone density loss,bone mass loss,osteoporosis and tooth loss caused by periodontitis,are in urgent need of bone regeneration to treat and repair bone defects.Therefore,understanding the osteogenic mechanism of bone marrow mesenchymal cells under the condition of inflammation and improving their biological properties under the influence of inflammation are one of the key factors to achieve periodontal tissue regeneration.The self-renewal and differentiation of stem cells are inseparable from their specific microenvironment.If the signals and components in the microenvironment are maladjusted,it will cause the disorder of self-renewal and differentiation of stem cells,and then lead to the occurrence of diseases.In the course of a variety of degenerative diseases,there are often immune changes and abnormal inflammatory factors,and the presence of excessive inflammatory factors often blocks endogenous stem cells-induced tissue regeneration.As one of the important factors of influencing stem cell nest steady-state inflammation can significantly affect the biological organism of internal environment stable,between effects include the function of the various types of stem cells,mesenchymal stem cells and biological function of neighboring cells can be affected,and then to a variety of negative regulation of tissue regeneration.As one of the adaptive responses to tissue injury,acute inflammation can promote the repair of injury.Normal tissue activates stem cells when it is damaged,and the activated stem cells play an important role in tissue repair.Acute inflammatory response is a typical humoral immune process,but in fact the role of stem cells in this process is often ignored.A typical example is that there are experiments to simulate the process of fracture healing.As one of the important cytokines in inflammatory response,TNF-? reaches its peak at 24 hours after fracture.TNF-? stimulated adipose-derived mesenchymal stem cells for a short period of time.High concentrations of TNF-? near fractures recruited and mobilized more stem cells to participate in tissue repair,thereby enhancing the osteogenic ability of mesenchymal stem cells.Compared with acute inflammatory response,chronic inflammation tends to disrupt local tissue reconstruction and defense,and the biological behavior of MSCs from different sources can be affected by TNF-?.The multidirectional differentiation ability of MSCs from periodontal tissue such as periodontal membrane was inhibited by TNF-?,which exacerbated alveolar bone defects caused by periodontal inflammation.TNF-? can cause pathological proliferation of gingival tissue-derived MSCs,and cause inflammatory gingival hyperplasia.Wnt signaling pathway generally includes classical Wnt/?-catenin signaling pathway and non-classical Wnt/Ca+signaling pathway,which is of great significance in bone tissue regeneration.Wnt/?-catenin signaling pathway supports osteogenic differentiation of bone marrow mesenchymal stem cells in chronic inflammatory microenvironment.GSK-3? is involved in the process of cell differentiation through different pathways.For example,GSK-3? affects the osteogenic differentiation of osteoblast cell lines and bone marrow mesenchymal stem cells through Wnt/?-catenin signaling pathway.The differentiation of bone marrow mesenchymal stem cells into cartilage is affected by NF-?B pathway.By inhibiting GSK-3? expression,mesenchymal cells with multidirectional differentiation potential could differentiate into osteoblasts.Silencing GSK-3? or phosphorylation and inactivation of GSK-3?reduced the degradation of ?-catenin and increased its concentration.Stably expressed?-catenin entered the nucleus to activate target genes and promote osteogenic differentiation of BMSCS.Therefore,understanding how the body reverses pathological damage and allows tissue repair and regeneration requires a deeper understanding of the complex interrelationship between stem cells and inflammation.MicroRNA is a kind of short endogenous single-stranded non-coding RNA.By inhibiting or degrading the mRNA of target gene after translation,microRNA plays an important regulatory role in a variety of physiological and pathological processes of cells,and is a hot research topic in target gene therapy in recent years.Studies have shown that miRNA can regulate the osteogenic differentiation of mesenchymal stem cells.In the early stage,miR-34a was mainly used for cancer inhibition.In recent years,scholars found that miR-34a not only inhibits the proliferation,growth and migration of various tumor cells,but also significantly inhibits the bone metastasis of cancer.Therefore,miR-34a has become a class of miRNA with more reports and clearer effects in bone metabolism in recent years.miR-34a is not only a natural osteoclast inhibitor,but also plays a complex regulatory role in the process of osteogenic differentiation.In addition,the relationship between miR-34a and inflammatory response,chondrogenesis,bone tissue repair response and tooth development has been gradually reported.miR-34a is not only involved in bone metabolism under physiological conditions,but also can regulate the biological behavior of osteoblasts under pathological conditions.miR-34a plays a regulatory role through a number of signaling pathways related to bone metabolism,such as the widely studied Wnt/?-catenin signaling pathway,Notch signaling pathway,MAPK(ERK1/2)signaling pathway,etc.However,the effect of miR-34a-5p on the specific biological behavior of rBMSCs in the inflammatory microenvironment is rarely reported.For a Long time,Long non coding RNA(LncRNAs)was considered as "dark matter",that is,an evolutionary garbage without biological function.However,in recent years,more and more evidences suggest that LncRNAs with a higher percentage of intracellular transcription can participate in many life processes and are important functional regulatory factors that cannot be ignored in cells,with more complex and important biological functions.LncRNA is closely related to many pathologic processes,especially inflammatory reactions.It has been confirmed that LncRNAs of MSCs osteogenic differentiation are involved in both normal and disease conditions.At the same time,some studies have confirmed that affecting the expression of LncRNA-NEAT1 can further cause inflammatory injury of periodontal tissue.Under the inflammatory microenvironment,BMSCs produce new LncRNAs,and there are a large number of differentially expressed LncRNAs,which affect the development and differentiation of mesenchymal stem cells.Some LncRNAs can play the role of ceRNA and form a regulatory network with miRNAs to regulate the protein coding of target genes.In this study,bioinformatics analysis previously found that some bases of LncRNA-NEAT1 and miR-34a-5p are complementary,so it was speculated that LncRNA-NEAT1 might regulate miR-34a-5p through sponging.The previous studies have confirmed that GSK-3? is the target gene of miR-34a-5p.Therefore,this study speculated that the LncRNA-NEAT1/miR-34a-5p/GSK-3? axis may affect the osteogenic differentiation of rBMSCs.To investigate the regulatory mechanism of NEAT1 and miR-34a-5p on osteogenic differentiation of mesenchymal stem cells through the overexpression or silencing of NEAT1 and miR-34a-5p.Based on the above,we intend to study the changes of miR-34a-5p in the process of bone remodeling in the inflammatory microenvironment,as well as the effect of miR-34a-5p on bone remodeling in the inflammatory microenvironment,and explore the possible mechanism of the effect of miR-34a-5p.Materials and Methods1.Rat bone marrow mesenchymal stem cells were routinely extracted,cultured and isolated,and then induced into osteogenic and adipogenic differentiation respectively.The expression of mesenchymal stem cell surface markers CD90,CD44 and CD29 and hematopoietic stem cell surface markers CD34 and CD45 were detected.2.The reversal effect of miR-34a-5p on TNF-? induced rBMSCs osteogenic inhibition was detected.Realtime PCR and Western blot were used to detect the expression of Runx2 and Osterix after TNF-? induction and miR-34a-5p intervention,ALP staining,alizarine red staining and quantitative analysis of mineralized nodules.3.Effect of miR-34a-5p/GSK-3? axis on osteogenic differentiation of rBMSCs induced by TNF-?.The changes of Wnt/?-catenin signaling pathway related proteins in the inflammatory microenvironment were detected through the overexpression of miR-34a-5p and the inhibition of miR-34a-5p,respectively.Luciferase reporter gene was used to detect the regulatory effect of miR-34a-5p on target gene GSK-3?.After inhibiting GSK-3? expression,the changes of osteogenesis of rBMSCs were detected by alizarin red staining and Western blot.4.Preliminary study of LncRN A-NEAT1 influencing rBMSCs osteogenic differentiation in inflammatory microenvironment through miR-34a-5p/GSK-3?axis.The expression level of lncRNA-NEAT1 after down-regulated miR-34a-5p and miR-34a-5p after down-regulated LncRNA-NEAT1 were observed by PCR.Results1.After conventional in vitro culture,the cells were passed by enzyme digestion method,and the cells were in good condition,grew rapidly,and the induction of osteogenesis and lipid formation was successful.Stem cell flow cytometry was used to detect positive expression of mesenchymal stem cell surface markers CD90,CD44 and CD29,and negative expression of hematopoietic stem cell surface markers CD34 and CD45.2.The osteogenic differentiation ability of rBMSCs is significantly reduced under TNF-? stimulation.After over expression of miR-34a-5p,the expression of osteoblast-related genes Runx2 and Osterix is up-regulated,and ALP and alizarine red staining are enhanced.After down-regulation of miR-34a-5p,the expression of osteoblast-related genes Runx2 and Osterix are down-regulated,and ALP and alizarin red staining are decreased.3.TNF-? inhibits the expression of miR-34a-5p through the NF-?B pathway.Combined with bioinformatics analysis,acquired/lost function test and luciferase reportingsystem,miR-34a-5p is confirmed to bind to GSK-3? mRNA 3'UTR to inhibit its expression.miR-34a-5p activates the Wnt/?-catenin signalingpathway by targeting GSK-3?.4.Bioinformatics analysis combined with luciferase reporter gene confirmed that LncRNA-NEAT1 and miR-34a-5p form a closed ring of mutual inhibition of expression.LncRNA-NEAT1 can negatively regulate the expression of GSK-3?.Under TNF-? stimulation,down-regulation of LncRNA-NEAT1 can promote the expression of osteoblast-related genes,increase ALP activity,and promote stem cells to form more bone nodules.On the contrary,the up-regulation of LncRNA-NEAT1 inhibits the expression of osteoblast-related genes,and reduces the activity of ALP and the ability to form osteogenic nodules.Conclusions1.TNF-? can inhibit rBMSCs osteogenic differentiation by affecting the expression of specific miRNA.Up-regulation of miR-34a-5p can antagonize the inhibitory effect of TNF-? on rBMSCs osteogenic differentiation,and partially restore rBMSCs osteogenic differentiation abilityin inflammatory microenvironment,suggesting that miRNA mediated post-transcriptional regulation plays an important role in rBMSCs osteogenic development under inflammatory conditions.2.miR-34a-5p promotes the osteogenic differentiation of rBMSCs stimulated by TNF-? by activating the Wnt/?-catenin signaling pathway,and partially reverses the decreased osteogenic differentiation of rBMSCs induced by TNF-?.LncRNA-NEAT1 and miR-34a-5p form a negative regulation closed loop,and both of them can affect the osteogenic differentiation ability of rBMSCs by regulating the expression of GSK-3?.