The Mechanism Of LncRNA MALAT1 Targeting The MiR-124-3p/IGF2BP1 Axis To Regulate Osteogenic Differentiation

The bacterial associated immune-mediated disease characterized by gingival bleeding,destruction of periodontal support tissue,and tooth loss.With the aging of the world population,periodontitis has become a serious public health problem affecting20-50% of the population.Plaque is the most common etiological factor and it is also affected by genetic and environmental factors.Traditional treatment methods include mechanical scaling and drug adjuvant therapy.It can lead to soft tissue trauma and drug resistance,which limits its efficacy and application.With the progress of modern medical research,it has been found that non coding RNA(nc RNA)is involved in the regulation of various biological processes,and it can play a role at the RNA level,rather than traditional coding proteins.Long non-coding RNA(lncRNA)is a class of RNA sequences longer than 200 nucleotides which have structural characteristics of m RNA.Lnc RNA itself does not encode proteins,but can regulate gene expression through multiple levels of epigenetics,transcription,and post transcriptional regulation.At present,multiple lncRNAs have been shown to regulate a variety of cellular processes in tooth-associated stem cells,such as osteogenic differentiation,inflammation,autophagy,proliferation,and apoptosis.Lnc RNA lung adenocarcinoma metastasis-associated transcript 1(lncRNA MALAT1)has become a research hotspot because of its rich biological functions.Lnc RNA MALAT1 can regulate gene transcription in tissue inflammation,tumor progression and cardiovascular remodeling.In recent years,lncRNA MALAT1 has been studied to play an important role in the regulation of osteogenesis in a variety of stem cells.Based on RNA sequencing studies,many lncRNAs are differentially expressed in the osteogenic differentiation process of PDLSCs and usually interact with miRNAs to regulate osteogenesis.The role of lncRNA MALAT1 in osteoporosis and related bone and joint diseases has been described one after another.The precise molecular pathways and specific biomarkers of periodontitis are still unclear,so exploring the molecular mechanisms of periodontitis is key to the development of targeted therapies in the future.PDLSCs are essential stem cell repositories in periodontal tissues which are relatively easy to obtain and have strong self-renewal and multi-differentiation potential.Therefore,whether lncRNA MALAT1 can regulate PDLSC osteogenesis through endogenous mechanisms becomes the focus of this study.In Chapter 2,PDLSCs were isolated and cultured from human teeth in vitro by tissue block combined with enzyme digestion method,and identified by flow cytometry,which proved that PDLSCs had the characteristics of mesenchymal stem cells and osteogenic differentiation was induced,and osteogenic-related markers increased significantly,indicating that PDLSCs successfully differentiated into osteoblasts.overMALAT1 and knockdown(si-MALAT1)plasmid which q RT-PCR proved that the construction was successful.PDLSCs were transfected with it respectively after osteogenesis induction.QRT-PCR and Western blot analyzed the levels of osteogenesis-related genes which revealed significant elevation within the overMALAT1 group.Fluorescence in situ hybridization showed that lncRNA MALAT1 was mainly located in the cytoplasm of PDLSCs,suggesting that lncRNA MALAT1 may regulate osteogenesis of PDLSCs through ce RNA mechanism.In Chapter 3,we predicted that lncRNA MALAT1 could target miR-124-3p through the bioinformatics website and confirmed it by dual luciferase experiment.Then,miR-124-3p mimic and inhibitor were transfected into PDLSCs respectively,and then osteogenic differentiation was induced.q RT-PCR and Western blot results showed that overexpression of miR-124-3p significantly inhibited osteogenic differentiation of PDLSCs.Alkaline phosphatase and alizarin red staining gave the same results.Combined with Chapter 2,lncRNA MALAT1 promoted osteogenic differentiation of PDLSCs,but miR-124-3p inhibited partial osteogenic differentiation.In Chapter 4,we predicted that IGF2BP1 could bind to miR-124-3p through the bioinformatics website and confirmed it through a dual luciferase experiment.To detect whether IGF-2BP-1 promoted the osteogenic differentiation of PDLSCs,we set two concentrations of 50ng/m L and 100ng/m L.q RT-PCR and Western blot results showed that IGF2BP1 could bind to miR-124-3p in an osteogenic induction medium.50ng/m L IGF2BP1 promoted osteogenic differentiation of PDLSCs more significantly than100ng/m L IGF2BP1.In the osteogenic induction medium,when si-MALAT1 was added,the osteogenic ability of PDLSCs decreased significantly,which was consistent with the results in Chapter 2.However,when IGF2BP1 was added at 50ng/m L,the osteogenic ability of PDLSCs increased,indicating that IGF2BP1 recovered part of the osteogenic inhibition mediated by si-MALAT1.In Chapter 5,we further verified whether lncRNA MALAT1 promoted skull regeneration in vivo.First,a skull defect model of SD rats was constructed.PDLSCs were transfected with si RNA(NC)and si-MALAT1 respectively,and then mixed with Matrigel glue 1:1.PDLSCS were placed on both sides of the skull defect area and killed8 weeks later.Three-dimensional reconstruction images of skull showed that bone regeneration in the defect area was inhibited after lncRNA MALAT1 was inhibited.Bone volume fraction(BV/TV)decreased.Masson and HE stainings showed that new bone formation decreased significantly.Combined with previous in vitro experiments,lncRNA MALAT1 was further demonstrated to be a positive regulator of bone regeneration.In conclusion,this study reveals that lncRNA MALAT1 regulates osteogenic differentiation of PDLSCs by targeting miR-124-3p/IGF2BP1 axis during osteogenic differentiation and has been verified in vitro and vivo experiments.This study provides potential therapeutic significance for periodontal tissue regeneration and bone defects in the future.