Mechanism Of LncRNA-Dnm3os Inhibiting Osteoblast Differentiation And Maturation

BackgroundBone can not only be used to support and protect the body,but also can cooperate with muscles for related support activities,movements,etc.,and can perform hematopoietic activities and store some minerals(such as calcium)[1,2].Although the bones appear to be static tissue,in reality,it is dynamic bone tissue that is continuously reabsorbed and formed,which is called "skeletal remodeling." Osteoclasts differentiated from hematopoietic stem cells are mainly responsible for bone resorption,while osteoblasts differentiated from bone marrow mesenchymal stem cells are mainly responsible for bone formation [1-5].The differentiation and function of these cells are strictly regulated and regulated by hormones and local cytokines.In normal bones,there is a balance between absorption and formation of bones,and the mass of bones remains a relatively constant value.However,in osteoporosis,rheumatoid arthritis(RA),and periodontitis,inflammatory bone-related diseases,the bone resorption capacity in the human body exceeds the bone formation capacity,so it is out of balance.Long non-coding RNAs(Lnc RNAs)are a group of RNAs greater than or equal to 200 nucleotides(nt)without or with very low protein coding restriction.In the human genome,14,470 have been identified 15787 lnc RNA transcripts of the lnc RNA gene [6,7].For a long time,lnc RNA was thought to be a byproduct of transcription of non-biologically functional RNA polymerase II.However,recent studies have found that lnc RNA plays a vital role in regulating nuclear chromatin structure and gene expression,and is also an active participant in disease development and development [8-10].A series of lnc RNAs have been confirmed to participate in the regulation of osteoblast differentiation and maturation.Despite recent advances in lnc RNA,the function of most lnc RNAs remains unclear.RUNX2 is essential for the osteogenic differentiation process and the regulation of related genes [11-13].RUNX2 is one of the three members of the RUNX family.It has a common conserved Runt domain and can promote communication with other proteins [14-17].All three members(Runx1,Runx2,and Runx3)play a pivotal role in skeletal development [18-20].Runx2's function is attributed to the protein structure encoded by its m RNA sequence.Runx2's various sites contribute to bone remodeling,and each of its unique functions is based on its interactions with other proteins and DNA sequences).The regulatory relationship between lnc RNA and RUNX2 is still under investigation.In this subject,we sequenced the lnc RNA of osteoblast precursor cells MC3T3-E1 and osteoblasts and sequenced them.We found that lnc RNA-Dnm3 os is a long-chain non-coding RNA with the largest expression difference in osteogenic differentiation.We further found that lnc RNA-Dnm3 os can inhibit MC3T3-E1 differentiation into osteoblasts in vitro.Inhibition of lnc RNA-Dnm3 os can promote osteoblast differentiation and maturation.We further found in the body.Injection of interfering RNA that inhibits lnc RNA-Dnm3 os can promote fracture healing.Finally,we conducted related mechanism research and found that inhibition of lnc RNA-Dnm3 os can promote the protein expression of RUNX2.So in the end we think that lnc RNA-Dnm3 os inhibits the differentiation and maturation of osteoblasts by inhibiting the protein expression of RUNX2,and further delays the fracture healing.ObjectiveLong-chain non-coding RNAs can play an active and effective role in cardiovascular and cerebrovascular diseases,immune system diseases,skeletal metabolic diseases,etc.,while lnc RNA-Dnm3 os can inhibit the maturation and differentiation of osteoblasts and bone formation.See the report.This project focuses on the research of Lnc RNA-Dnm3 os inhibiting osteoblast differentiation and mature cells,animal experiments,etc.,and its related mechanism.In this study,we first cultured MC3T3-E1 and osteoblasts.High-throughput sequencing was performed to build a long-chain non-coding RNA to screen out differential lnc RNA-Dnm3 os.Secondly,we verified that it can promote the differentiation and maturation of osteoblasts at the cell level by interfering with lnc RNA-Dnm3 os,and in fractures In the model,it was verified whether lnc RNA-Dnm3 os can delay the healing of fractures.Finally,molecular interactions such as Western-blot and real-time fluorescent PCR were used to verify the relationship between RUNX2 and RUNX2.Part I: Screening and identification of key long-chain non-coding RNAsMethods:(1)Cultured osteoblast precursor cell lines and osteoblasts 21 days after cultured in osteogenic induction medium,total RNA was extracted and subjected to full transcriptome sequencing analysis.(2)Collect the total RNA of osteoblast precursor cells and osteoblasts,and detect the expression of key lnc RNA and the expression of osteogenic marker differentiation genes by reverse transcription and q PCR.RESULTS: The content of lnc RNA Dnm3 os in osteoblasts was reduced and screened by the full transcriptome sequencing analysis.The content of lnc RNA Dnm3 os was significantly decreased in osteoblasts after q RT-PCR.Learn the difference.Conclusion: lnc RNA Dnm3 os plays a key role in the differentiation of osteoblasts.Part II: Lnc RNA Dnm3 os inhibits osteoblast differentiation and bone formationMethods:(1)MC3T3-E1 cell line was cultured and transfected with lnc RNA Dnm3os-si RNA.The maturation of osteoblasts was observed by differentiation into osteoblasts and alizarin red staining.(2)The expression level of osteogenic related marker genes was detected by q PCR.(3)Fracture mouse models were constructed and randomly divided into two groups.One group was injected with lnc RNA Dnm3os-si RNA in the tail vein,and the other group was injected with the negative control si RNA,and the bone fracture recovery was observed by HE staining and micro-CT.Results: After differentiation into osteoblasts by MC3T3-E1,alizarin red staining and q PCR detection revealed that si RNA osteoblasts transfected with lnc RNA Dnm3 os increased significantly.A mouse model of fracture was constructed in vitro.The mouse group injected with lnc RNA Dnm3os-si RNA in the tail vein had significantly faster fracture healing than the control group.The same phenomenon was observed in H-E staining and ALP staining.Conclusion: lnc RNA Dnm3 os can inhibit osteoblast differentiation in vitro.The group of fracture mice injected with lnc RNA Dnm3os-si RNA in the tail vein healed faster than the control group.Part III: mechanism of lnc RNA Dnm3 os inhibiting osteoblast differentiation and maturationMethods:(1)Extract BMSCs from mice injected with lnc RNA Dnm3os-si RNA in the tail vein and osteogenicly differentiate into osteoblasts.Alizarin red staining was used to observe its morphology and calcium deposition.(2)Through protein imprinting experiments(WB),observe changes in the expression level of osteogenic related proteins.(3)According to the STARBASE database,mi RNAs competing with lnc RNA Dnm3 os were found.(4)The direct interaction between lnc RNA Dnm3 os and mi R-328 was verified by luciferase report experiment.(5)Overexpression or inhibition of mi R-328.Observe the change of the number of osteoblasts when MC3T3-E1 differentiates into osteogenesisResults:(1)Observed the effect of lnc RNA Dnm3os-si RNA on the differentiation of osteoblasts in primary cell BMSCs;(2)WB experiments showed that lnc RNA Dnm3os-si RNA transfected in vitro increased the expression of RUNX2.(3)Through the STARBASE database,it was found that lnc RNA Dnm3 os may have a ce RNA competition relationship with mi R-328.(4)Through the luciferin report experiment,it was found that there is a direct competition between lnc RNA Dnm3 os and mi R-328.(5)Overexpression of mi R-328 promotes MC3T3-E1 differentiation into osteogenesis,while inhibiting mi R-328 inhibits MC3T3-E1 differentiation into osteogenesis.Conclusion:(1)lnc RNA Dnm3 os and mi R-328 are endogenous ce RNA;(2)lnc RNA Dnm3 os inhibits MC3T3-E1 differentiation into osteogenesis by competing for mi R-328 binding.