The Effect Of Long Noncoding RNA TUG1 On The Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells And The Underlying Mechanism

Background and ObjectivesPeriodontal tissue regeneration is one of the focuses of oral medicine research,which is the common goal of multidisciplinary field research in periodontology,maxillofacial surgery,orthodontics and prosthodontics.In recent years,the research progress of dental stem cell and tissue engineering technology has brought new research direction to the biological regeneration of periodontal tissue.Periodontal ligaments stem cells(PDLSCs)are a group of mesenchymal stem cells derived from periodontal ligament tissue that are easily available in oral clinical,also a kind of heterogeneous pluripotent stem cells with physiological self-replication ability and multi-directional differentiation potential.PDLSCs which were successfully isolated and cultured since 2004,have been attracted the attention of researchers in the field of stomatology and other biological regeneration,and have been defined as promising alternative cell sources for repairing missing tissues.They exerted their potentials to differentiate into a wide variety of cell types,particularly differentiate into osteoblasts to form the cementum-periodontal ligament-alveolar like structures and rebuild the attachment relationship.Diverse evidences also confirmed that the fully understanding of the regulatory mechanism of PDLSCs osteogenic differentiation was prerequisite to achieve and improve in therapeutic approaches,and also served as a stepping stone for the development of novel therapeutic strategies that can be used to regenerate dental tissues.Long noncoding RNAs(lncRNAs)are defined as transcripts longer than 200 nucleotides in mammalian genomic transcriptional activity.They originally regarded as transcriptional "noise" and had no biological function.However,with the extensive application of the second-generation gene sequencing technology and the specific research,lncRNAs have been found to play important roles in many biological fields,such as stem cell biology,X chromosome inactivation,individual development,tissue formation and metabolism.Growing evidences indicated that lncRNAs have emerged as key modulators to participate in various physiological and pathological processes,which can regulate the related genes expression from epigenetic,transcriptional and post-transcriptional levels in human diseases.In pluripotent stem cells,there are evidences that the potential roles of some specific lncRNAs could affect a wide range of cellular activities including cell differentiation,self-renewal,proliferation,apoptosis,etc.For example,HoxA-AS3 acted as an epigenetic switch to associate with Enhancer of Zeste 2(EZH2)and repressed the transcription of key osteoblastic factor(Runx2)to regulate the osteogenic differentiation of bone marrow mesenchymal stem cells;lncRNA-POIR may act as a competing endogenous(ceRNA)for miRNA-182 to adjust the expression of FoxO1 in the process of osteogenic differentiation of inflammatory periodontal ligament stem cells,and provide new therapeutic strategies and targets for the treatment of periodontitis.In previous study,we found that miRNA-21 could regulate the osteogenic differentiation of PDLSCs by targeting SMAD family member 5(Smad5)in vitro and in vivo.With regard to the potential relationship of miRNA and IncRNA,we searched IncRNAs that were correlative to miRNA-21 and selected three functional IncRNAs for validation.Taurine upregulated gene 1(TUG1)was one of the related IncRNAs,and the expression of TUG1 was most obviously changed in osteogenic differentiated PDLSCs.LncRNA-TUG1 is approximately 7.1 kb in length and widely present lncRNA in human body.It was initially identified as an essential gene for retinal development and forming photoreceptors in mouse eye,was expressed in the nucleus and in the cytoplasm.Silencing the expression of IncRNA-TUG1 in early retina cells would inhibit cell growth or increase apoptosis,causing photoreceptor blockade and retinal developmental deformity.Subsequent studies are mainly reflected that lncRNA-TUG1 can promote various tumor cell growth,proliferation,cell programmed death,epithelial mesenchymal transformation,migration and cell signaling ability in human non-small cell lung cancer(NSCLC),invasive bladder cancer and esophageal squamous cell carcinoma(ESCC).Additional reports indicated that IncRNA-TUG1 can be combined with PRC2 protein complex to precise regulate the expression of the growth control genes.However,the role of lncRNA-TUG1 on the osteogenic differentiation of PDLSCs and its mechanism has never been explored.In this study,we first isolated PDLSCs for osteogenic induction and assessed the potential of cells to differentiate into osteoblasts.According to the research of miRNA-21 and bioinformatics methods,combining with the regulation mechanism of lncRNA as "miRNA bait" in cellular activities,we screened out IncRNA-TUG1 the expression of which had obviously changed during PDLSCs osteogenic differentiation.To investigate the function of IncRNA-TUG1 in the process of PDLSCs osteogenic differentiation,we constructed a stable cell model of silencing lncRNA-TUG1 by lentiviral vector and analyzed the expression of osteogenic-related markers in osteogenically induced PDLSCs compared to their undifferentiated counterparts.Then,we explored the potential target protein of lncRNA-TUG1 on the osteogenic differentiation of PDLSCs by screening techniques and probed the relationship between them.Finally,clarified the molecular mechanism by which lncRNA-TUG1 regulates osteogenic differentiation of periodontal ligament stem cells.Our results provided novel insights into the mechanism that underlies the osteogenic differentiation of PDLSCs and could serve as a stepping stone for the development of novel therapeutic strategies that can be used to regenerate dental tissues.Materials and Methods1.Isolatied and identified PDLSCs.The classical method(tissue block combined with enzyme digestion method)was used to isolate and culture human periodontal ligament stem cells in vitro.Primary PDLSCs were observed morphologically and drawn the growth curves.Flow cytometry and immunofluorescence were used to identify the stem cell surface markers.The potential of osteogenic or adipogenic differentiation was detected by ALP staining,Alizarin red staining,Oil red O staining and qRT-PCR.2.Screening the differentially expressed IncRNAs and determining IncRNA-TUG1 during PDLSCs osteogenesis differentiation.According to the research of miRNA-21 regulating PDLSCs osteogenic differentiation,Starbase V2.0 software was used to predict the related IncRNAs of miRNA-21.PDLSCs were selected and cultured with osteogenic induction medium for 7 days.The special lncRNA(TUG1)was identified by qRT-PCR.To further investigate whether lncRNA-TUG1 affects the osteogenic differentiation of PDLSCs,we assayed the subcellular localization of lncRNA-TUG1 and the dynamic expression profiles.3.Construction of the stably transfected PDLSCs with silencing IncRNA-TUG1 and evaluation the functional of lncRNA-TUG1 on the osteogenic differentiation of PDLSCs.The recombinant lentiviruses of human TUG1 knockdown were designed and constructed by Link Biotech(Shanghai,China).The empty vector was served as a negative control.According to the requirements,we first conducted the preliminarily experiment to obtain optimum value of multiplicity of infection(MOI)and screen out the most effective vector.The silencing lncRNA-TUG1 of PDLSCs model was got.In the formal experiment,we used several classical approaches to evaluate the biological effects of IncRNA-TUG1 in the osteogenic differentiation of PDLSCs.The detection of ALP activity was determined from two respects(qualitative analysis and quantitative analysis)by ALP staining and ALP quantification after inducing different times.After 3 weeks of ossification induction,Alizarin red staining and quantitative calcium measurements were used to evaluate the mineralization of PDLSCs calcium matrix.The expressions of osteoblast-related genes ALP,Runx2 and OCN were detected by qRT-PCR.4.Predicting the target protein of IncRNA-TUG1 and exploring the underlying mechanism of lncRNA-TUG1 during PDLSCs osteogenic differentiation.Through with bioinformatics prediction,the potential target protein of lncRNA-TUG1 was explored in the process of osteogenic differentiation of PDLSCs.We analyzed the subcellular localization of potential target proteins by cytoplasmic and nuclear isolation methods;and determined the regulation role of IncRNA-TUG1 by qRT-PCR,Western blot and other screening techniques.In order to further detect the effect of target protein on osteogenic differentiation of PDLSCs,we clarified the relationship of lncRNA-TUG1 and target protein,and observed the expression of IncRNA-TUG1 and osteoblast-related genes by small interfering RNA of target protein.Results1.Primary PDLSCs were isolated through with tissue block combined with enzyme digestion method.Cells successfully exhibited fibroblast-like and radial growth morphology in culturing condition.The passaged cells showed a thriving growth and expressed mesenchymal stem cell markers(STRO-1 and CD 146);but were negative for leucocyte cell maker(CD45)and platelet endothelial cell marker(CD31).Next,we confirmed that our PDLSCs had pluripotency when cells were cultivated in osteogenic or adipogenic differentiation induction medium.2.Combined with the previous report of miRNA-21,19 putative IncRNAs were predicted by bioinformatics.After osteogenic inducing of PDLSCs for 7 days,we identified three functional lncRNAs(including TUG1,SNHG1 and XIST)and verified lncRNA-TUG1 changed most significantly between the non-induced group and the osteogenic-induced group.In addition,the expression of IncRNA-TUG1 was increased gradually in a time-dependent manner at Od,1d,3d and 7d after osteogenic induction;and decreased at 10d and 14d,but still increased compared with the control group.The results of subcellular distribution also showed that lncRNA-TUG1 was broadly localized in the cells but showed a significantly higher level in the nucleus than cytoplasm.3.In silencing experiment,PDLSCs were transfected by lentivirus.The transfection efficiency of them was very high after 72 h transfecting.The stable and translucent green fluorescence confirmed that the virus vector with the target sequence was stably transfected into cell.qRT-PCR suggested that sh-TUG1-2#had the lowest level of endogenous lncRNA-TUG1 expression.Subsequent osteogenic cultured,ALP activity,ALP staining,Alizarin red staining,calcium ion concentration and the mRNA levels of osteogenic genes(ALP,Runx2 and OCN)in silencing group were significantly lower than others.These results strongly suggested that lncRNA-TUG1 was involved in regulating the osteogenic differentiation of PDLSCs.4.We scanned Starbase V2.0 for potential target proteins of lncRNA-TUG1 and identified 28 putative candidates from different cell types.Then,we focused on 10 RBPs that have been reported to play regulatory roles in the differentiation of stem cells or have the relationship of lncRNA.qRT-PCR analysis indicated that the mRNA transcripts of PUM2,IGF2BP1,IGF2BP2,IGF2BP3,Lin28A and TNRC6A were preferentially localized in the nucleus.Among these candidates,Lin28A showed the highest transcriptional level in the nucleus and thus was selected for further analysis.Gene co-expression network analysis indicated that there were multiple binding sites and existed favorable hybridization energy(-11.8782 kcal/mol)between Lin28A and lncRNA-TUG1.Furthermore,Western blotting found that the protein level of Lin28A declined significantly in TUG1-repressed PDLSCs compared to the un-transfected cells.Taken together,these results suggested a correlation between the level of lncRNA-TUG1 and that of Lin28A during the osteogenic differentiation of PDLSCs.On the other hand,we confirmed the mRNA and protein levels of Lin28A by siRNA.Subsequent knockdown of Lin28A with si-Lin28A-2#not only led to a significant reduction in the expression level of lncRNA-TUG1,but was also shown to lower the osteogenic potential of the transfected PDLSCs.These data implied that Lin28A could be implicated in the modulation of osteogenic differentiation in PDLSCs.Conclusions1.LncRNA-TUG1 is widely distributed in human periodontal ligament stem cells.The expression of IncRNA-TUG1 is upregulated in the process of osteogenic differentiation of PDLSCs.2.Knockdown of IncRNA-TUG1 could reduce the ALP activity of PDLSCs,inhibit the deposition and mineralization of calcium ions of cells,and decrease the expression of osteogenic genes.3.Lin28A,a target RNA binding protein of IncRNA-TUG1,would affect PDLSCs osteogenic differentiation.It could interact with lncRNA-TUG1 to form a positive regulatory network of RBP-lncRNA,and synergistically promote the expression of osteogenic-related genes in PDLSCs,and thus achieve the effect of IncRNA-TUGI in promoting the osteogenic differentiation of PDLSCs.Features and innovationThis study explored the dynamic expression of IncRNA-TUG1 in the process of PDLSCs osteogenic differentiation from cellular and molecular level.We used the interfering method and functional experiments to confirm that IncRNA-TUG1 was positively correlated with the osteogenic differentiation of PDLSCs following the induction.Additionally,we found that lncRNA-TUG1 and Lin28A(the target RBP)formed a lncRNA-RBP network and promote the osteogenic differentiation of PDLSCs.Taken together,our study firstly explored the regulatory mechanism of PDLSCs osteogenic differentiation from lncRNA and RBP,help us better understand the mechanism that governs the osteogenic differentiation of PDLSCs,and also serve as a stepping stone for the development of novel therapeutic strategies that can be used to regenerate dental tissues.