The Screening And Identification Of LncRNAs Involved In Osteogenic Differentiation Of Human Periodontal Stem Cells

Background and objection:It is generally accepted that the function and preservation of the tooth depends on periodontium. The periodontal ligament （PDL）, a dense connective tissue bonding the roots of the teeth and alveolar bone, covering the roots can renew and repair itself, but this ability is limited. The reconstruction of periodontal tissue defects caused by periodontal diseases is the primary goal of periodontal therapy. Human periodontal ligament stem cells （hPDLSCs） are pluripotent stem cells with self renewal capacity and multiple differentiation potential. hPDLSCs can be easily acquired from PDL, and has become the fundamental elements for the repair of periodontal defects and gene therapy.Long non-coding RNAs （lncRNAs） are generally defined as RNAs longer than 200 nucleotides that do not code proteins, but nevertheless are functionally important. LncRNAs can act as regulators by influencing chromatin-modification, gene expression and translation process. It has become increasing clear that lncRNAs are key regulators of diverse processes, such as embryonic development, histological differentiation and organ formation.Specific expression character of IncRNAs has be functionally associated with the devefopment of many complex diseases. And it has been reported that IncRNAs play important roles in the maintenance of stem cell pluripotency and multi-directional differentiation. Although few IncRNAs have been identified in hPDLSCs, it is possible that they might participate in the osteogenic differentiation of hPDLSCs. This study would be significant for revealing the important role of lncRNA in the reconstruction of periodontal tissue defects, and it also provides a new idea for periodontal tissue regeneration in the future.The thesis is composed of the following four chaptersChapter 1 The isolation and identification of hPDLSCsThe hPDLSCs used in this study were obtained using modified tissue explant collagenase method and limited dilution technique. The growth rate of hPDLSCs was examined using CCK8 method. Stem cell surface markers were analyzed by flow cytometry. Clone formation assay, osteogenic and adipogenic differentiation induction were performed to examine the colony forming capacity and multilineage differentiation capability.Chapter 2Microarray analysis and qRT-PCR verification of gene expression during osteogenic differentiation of hPDLSCsThe RNA extracted from experimental （osteogenic differentiation induction group） and control groups were analysed by microarray. Approximately 30,586 IncRNAs and 26,109 coding transcripts were detected. Quantitative reverse transcription polymerase chain reaction （qRT-PCR） was performed using a commercially available assay kit according to the manufacturer’s instructions.Chapter 3CNC analysis and selection of key lncRNA during osteogenic differentiationThe data of gene chip was briefly analyzed using gene ontology （GO） and pathway analyses. A Coding-non-coding gene co-expression （CNC） network was built to identify interactions among lncRNAs and mRNAs according to correlation analysis. The key lncRNA during osteogenic differentiation of hPDLSCs was selected according to the analyses. And the expression patterns of key lnRNA in hPDLSCs and PDL were verified using qRT-PCR method.Chapter 4The effect of TCONS₀0007046 suppression on osteogenic differentiation andproliferation of hPDLSCsqRT-PCR was used to verify the inhibition efficiency of TCONS₀0007046 shRNA lentivirus. The ability of cell proliferation was detected using CCK8 kit. Alkaline phosphatase （ALP） activity kit and alizarin red staining detected the osteogenic differentiation ability of shRNA-Lnc and shRNA-Ctrl hPDLSCs. qRT-PCR was used to reveal the expression levels of osteogenic differentiation related genes （ALP, OCN, Runx2, BMP2 and BMP6）.Materials and methodsSample collection and cell cultureHealthy impacted third molars were collected from individuals aged 18-25 years old at the Department of oral and maxillofacial surgery of Nanfeng Hospital, following approved guidelines set by the Ethical Committee of Southern Medical University. The subjects included in this study had no history of systemic disease, smoking, or special medicatioa The teeth were preserved in a-MEM culture solution containing penicillin and streptomycin right after extraction.PDL tissue was isolated from the middle 1/3 of the root surface and then washed with sterile PBS several times..The PDL tissue was minced to 1 mmxl mmxl mm size, and was digested in a solution of 3 mg/mL collagenase type I and 4 mg/mL dispase for 30 min at 37℃, followed by centrifugation at 800 rpm for 3 min. The cells and tissues were seeded into 6-well plate. And slides were placed to make sure the tissues would stick to the plate firmly. The explants were cultured in a-modified Eagle’s medium supplemented with 10% fetal calf serum,100μmol/L ascorbic acid 2-phosphate,2 mmol/L glutamine,100 U/mL penicillin, and 100μg/mL streptomycin, at 37℃ in 5% carbon dioxide. And the medium was changed every 2～3 days.Periodontal ligament cells at 1st passage were selected, and a limited dilution technique was used to obtain single cell-derived colonies. Briefly, the multiple proportion dilution method was performed to modulate cell density to 10-15 cell/mL. The cells were then seeded into 96-well plate with a volume of 100 μL/well. The medium of single-celled well was supplemented to 20 μL/well after cultured for 12 h. The medium was changed every 2-3 days. After reached 70～80% confluence, single cell-derived colonies were sub-cultured using Trypsin-EDTA digestion.Cell proliferation assay by CCK8For cell proliferation assay, hPDLSCs at 3rd passage were selected and seeded into 96-well plate at the density of 2×103 cells/well （a volumn of 100 μL/well）, and then cultured at 37℃ in 5% carbon dioxide. After attachment,10μL of CCK8 solution was added to each well. After incubation for 1-4 h away from light, the absorbance of each well was measured by a microculture plate reader with a test wavelength of 490 nm. The growth curve was drawn according to the absorbance at each time point.Flow cytometry analysis of stem cell surface markersAt least 1×106 cells at the 3rd passage were prepared in cold PBS for each test. hPDLSCs incubated with phycoerythrin （PE） conjugated monoclonal antibodies for humann CD146, CD 29, CD31, CD34, CD90 andallophycocyanin （APC） conjugated monoctonal antibodies for human Stro-1 were incubated at 4℃ away from light for 1 h. After washed with PBS cell phenotype analysis was performed by flow cytometric analysis according to the manufacturers’instructions.plate clone formation assayFor clone formation assay, hPDLSCs at 3rd passage were seeded on 100-mm culture dishes at a density of 1×102 cells/plate. The cells were then fixed with 4% paraformaldehyde and stained with crystal violet stain after cultured for 14 days. The number of colony-forming units （CFUs） was counted under microscope （50 or more cells were defined as a clone）.Induction of osteogenic and adipogenic differentiationhPDLSCs at the 3rd passage were seeded into 6-well plates at a density of 1×105 cells/well. Osteogenic differentiation induction medium or adipogenic medium was added when cells reached 80% confluency. The medium was changed every 3 days. And after cultured for 21 days, calcium accumulation and lipid droplets were observed after by staining with 2% alizarin red S and oil red O solution, respectively.RNA extraction and microarray analysisThree osteogenic-inducted cell samples were regarded as the experimental group, and three undifferentiated cell samples were regarded as the control group. After cultured for 14 days, total RNA was extracted from hPDLSCs using Trizol Reagent. The purity and concentration of RNA samples was evaluated using a NanoDropND-1000 ultraviolet spectrophotometer. The RNA extracted from experimental and control groups were analysed by microarray.Quantitative reverse transcription poly merase chain reaction （qRT-PCR）Total RNA was extracte from hDPSCs using Trizol Reagent. After cDNA was generated, the cDNA was amplified using SYBR（?） Premix DimerEraserTM according to the manufacturer’s instructions. GAPDH was used as an internal control, and relative gene expression was represented as fold change using the 2-ΔΔCt method.Bioinformatics analysesThe data of gene chip was briefly analyzed using clustering, gene ontology （GO） and pathway analyses according to databases, such as NCBI RefSeq, UCSC, RNAdb and LncRNAs. P value was used to define the threshold of significance （P< 0.05 was considered statistically significant）.Coding-non-coding gene co-expression （CNC） networkA CNC network was built to identify the interactions among lncRNAs and mRNAs during osteogenic differentiation in hPDLSCs according to correlation analysis. Pearson correlation analysis was applied based on the normalized signal intensity of individual genes. And CNC networks were drawn using the open source software Cytoscape.Construction of TCONS₀0007046 shRNA lentivirus and cell infectionThe oligonucleotides were cloned into GV118-GFP to generate the lentiviral vectors. And HEK293T was chosen to be the packaging cells （GeneChem, Shanghai, China）. The negative control used in this study contained no homology to any human gene. Briefly, the recombinant lentiviral vectors were transfected into 293T cells together with and packaging vectors. The concentrated lentivirus were collected after purified by ultracentrifugation and then the titer of lentiviruses was tested. hPDLSCs were infected with TCONS₀0007046 shRNA entivirus at different multiplicity of infection （MOI）. Expressions of green fluorescent were observed after 72 h culturing.Statistical analysisAll statistical analyses were performed using SPSS 19.0 software.All quantitative data were presented as mean±standard deviation（x±s）. Student’s t-test was used to determine the statistical significance. The association of the two variables was calculated using the Pearson product-moment correlation coefficient （r）. P< 0.05 was considered statistically significant.Results1. The cell p he no type characteristics and biological property of hPDLSCsThe hPDLSCs used in this study were obtained using modified tissue explant collagenase method and limited dilution technique. Their multipotency was evaluated by staining with alizarin red and oil red O confirmed their multipotency after 21 days of induction.The results of plate clone formation assay showed the clone formation ability. The flow cytometry analysis showed the hPDLSCs could express mesenchymal stem cell markers CD29 （99.62%）,CD90 （97.13%）, CD146 （10.27%）, and Stro-1 （12.76%）; but were negative for hematopoietic cell markers including CD31 （0.07%） and CD34 （0.22%）.2. Expression profiles of IncRNA and mRNA during osteogenic differentiationThe lncRNA and mRNA expression profiles of hPDLSCs during osteogenic differentiation was obtained by microarray analyses. The results of clustering analysis demonstrated that, after osteogenic induction,994 lncRNAs were up-regulated and 1177 lncRNAs were down-regulated; and 3557 mRNAs were identified as differentially expressed after osteogenic induction in hPDLSCs, of which,1578 were up-regulated and 1979 were down-regulated （fold change> 2.0 or<-2.0, P< 0.05）. qRT-PCR was used to validate the expression of a number of candidate lncRNAs and mRNAs, and the results suggested high consistency between qRT-PCR and microarray data.3. LncRNA-mRNA CNC network and selection of key lncRNAGO analysis indicated that differentially expressed mRNAs were related to many significant bio logical processes, including the categories cellular process, biological regulation, etc. The results of pathway analysis showed that 83 pathways were involved in differentiation, including the MAPK, VEGF, and TGF-beta signaling pathways. Several mRNAs reported to be related to osteogenic differentiation were chosen as targets （ALP, BMP6, COL1A1, COL1A2, IL6, BMP5 and BMP2）. And then the lncRNA-mRNA co-expression network was constructed to identify interactions among differentially expressed lncRNAs and mRNAs. The CNC network included 131 negative pairs and 262 positive pairs. LncRNA TCONS₀0007046 was selected as the key regulator based on the analyses and results, and its expression patterns in hPDLSCs and PLD were verified by qRT-PCR.4. The effect of TCONS₀0007046 suppression on osteogenic differentiation and proliferation of hPDLSCsThe results of qRT-PCR indicated that the TCONS₀0007046 shRNA lentivirus was constructed successfully. lncRNA TCONS₀0007046 has no obvious affect on the growth rate of hPDLSCs.The expression of TCONS₀0007046 was significantly increased after osteogenic induction in hPDLSCs. The results of ALP activity test showed that the ALP activity level was lower in the shRNA-Lnc hPDLSCs. The auzarin red staining indicated that mineralized nodules were fewere in the shRNA-Lnc hPDLSCs after 14 days’osteogenic induction.When compared to shRNA-Ctrl group, the expression levels of ALP, OCN, Runx2, BMP2 and BMP6 were down-regulated in shRNA-Lnc hPDLSCS after 14 days’osteogenisis.ConclusionIn summary, the hPDLSCs we successfully isolated using a limited dilution technique are clonogenic and capable of osteogenesis and adipogenesis in vitro; the results of microarray showed that the expression levels of IncRNAs before and after osteogenic differentiation in hPDLSCs were significantly different; The remarkable differences between the samples together with bio informatics analyses indicated that lncRNAs might play significant roles in the differentiation of hPDLSCs. LncRNA TCONS₀0007046 had no effect on the proliferation of hPDLSCS. When the expression of TCONS₀0007046 was inhibited, the ability of osteogesis of hPDLCs was somehow disturbed. And this regulation process might be related to the expression of osteogenisis-related mRNAALP, OCN, Runx2, BMP2 and BMP6.