Hypoxia Induces HIF-1α To Inhibit Biological Activity Of HPDLCs And Screening LncRNA Differentially Expressed In Periodontitis Tissues

Periodontitis,a kind of infectious disease,is caused by Gram-negative bacteria accumulating on the surface of teeth to form plaque,causing microcirculation changes in periodontal tissues,activating host cells to release inflammatory factors,leading to destruction of periodontal tissues,formation of gingival inflammation,periodontal pockets and alveolar bone absorption,finally leading to the teeth lose.The proportion of middle-aged residents in China with periodontal diseases is as high as 95%.Periodontitis not only affects the occurrence and development of multiple systemic diseases,but also has a serious negative impact on people’s mental health.Thus,early prevention and treatment of periodontitis is particularly important.In the current era of individualized diagnosis and treatment,despite the multidisciplinary efforts of genomics,transcriptomics,proteomics/polypeptides and metabolomics,there are still no biomarkers available for the diagnosis,prognosis and therapeutic evaluation for periodontitis.The exploration of these markers and the discovery of drug targets will help to improve the clinical evaluation and treatments ofperiodontitis.Tissue hypoxia is a pathological feature,mainly due to the disruption of cellular oxygen consumption and blood supply oxygen balance.The oxygen pressure of normal tissue is about 2.5%-9%,while it is 1%-2% in the wound and infected tissue sites.According to the reports,the inflammatory factors IL-18 and HIF-1α were significantly induced in hypoxia related periodontitis,indicating that HIF-1α plays a role in hypoxia related periodontitis.However,to our present knowledge,there are no reports focusing on the molecular mechanisms of how hypoxia affects the biological behaviors of human periodontal ligament cells(hPDLCs).LncRNAs are a group of non-coding RNA of approximately 200 nucleotides in length,which not only positively or negatively regulate the expression of coding genes through transcriptional interference or induction of chromatin remodeling,but also form sponge with miRNAs to promote the expression of miRNA downstream genes.They mainly participate in the regulation of various biological behaviors of cells during physiological and pathological processes.Hua Li et al.used rat transcriptome microarray 1.0 to detect abnormally expressed LncRNAs in a rat model of acute myocardial infarction caused by ischemia and hypoxia,and found 168 highly expressed LncRNAs and 155 lowly expressed LncRNAs.Further GO analysis and signaling pathways analysis revealed that aberrantly expressed LncRNAs are particularly involved in apoptosis,ERS and autophagy by regulating the PI3K-AKT signaling pathway.It is reported that LncRNA HOTTIP is highly expressed in hypoxic glioma cells,which promotes the expression of its downstream target gene ZEB1 by forming a sponge with miR-101,thereby accelerating the transfer and invasion ability of glioma cells in a hypoxic environment.The expression of LncRNA HAS2-AS1 was also significantly increased in oral squamous cell carcinoma cells(OSCCs)under hypoxic conditions,suggesting that hypoxia-induced LncRNAs may play a very important molecular role in tumorigenesis and inflammatory diseases through HIF-1α.However,at present,we are still unclear about LncRNAs expression modes in periodontitis samples.In this study,we isolated hPDLC in vitro,observed the changes of biological behavior and related signaling pathways of hPDLCs under hypoxic conditions,and detected differentially expressed LncRNA in gingival tissues of periodontitis by using LncRNA chip,which provided a theoretical basis for further exploring the deep molecular mechanism of the occurrence and development of periodontitis,molecular diagnosis of periodontitis and personalized targeted drug therapy.This study is divided into the following four parts:Part 1 Culture and identification of human periodontal ligament cells Objective: Isolate and culture hPDLCs in vitro and identify the cells forsubsequent experiments.Method: hPDLCs were isolated and cultured in vitro by tissue block method.Immunocytochemistry was used to identify hPDLC cells by vimentin and keratin staining.Results: We isolated and cultured 20 patients’ hPDLCs during the operation.During the process,hPDLCs of 6 patients were successfully cultured in vitro,about 30% for the primary culture rate.We found that hPDLCs from young people are much more easily isolated than the older people.During the culture process,the growth rate and state of hPDLC cells were significantly improved after 3passages.Moreover,the state of hPDLCs was better when cultured in complete DMEM media with 20% FBS,greatly improving the successful rate for the primary culture.The expotentially grown hPDLCs are large in volume,rich in cytoplasm,and cells are long fusiform or star-shaped when observed under the inverted microscope.However,when the cells were passaged to the 8th generation,the growth rate of the cells was significantly slowed down,the cell volume was more enlarged.What’s more,a lot of black particles began to appear in the cytoplasm.The cell adherence ability was significantly weakened after trypsin digestion,suggesting that the cells entered the aging stage.Subsequently,immunocytochemical staining of vimentin and keratin in hPDLC cells showed that the expression of vimentin was positive and mainly localized in the plasma of hPDLCs,while the keratin staining was negative,consisting with the reports that the hPDCLs are originated from mesoderm mesenchymal fibroblasts.Conclusion: hPDLCs from mesenchymal sources were successfully cultured in this experiment.Part 2 Changes of biological behavior of hPDLCs in hypoxic microenvironmentObjective: To observe the effect of hypoxia on the biological behavior of hPDLCs.Method: HPDLC cells in logarithmic growth phase were cultured in cell culture chambers with different oxygen concentrations(21%,5%,1%).MTT assay was used to detect cell proliferation,inverted microscopy was used to observe cell morphology and cell scratch test was used to detect cell migration.Results: After 14 days of incubation in different oxygen concentration incubators,the number of hPDLC cells was significantly reduced and arranged sparsely under one microscope.The structure of hPDLC changed obviously and tended to be senile.Scratch test data show that severe hypoxia can slow down the migration of hPDLCs.Conclusion: Hypoxia can inhibit the proliferation and migration of hPDLCs.Part 3 Changes of HIF1α and inflammation-related signaling pathways in hPDLCs under hypoxic microenvironmentObjective: To investigate the underlying molecular mechanisms of hypoxia on the biological behavior of hPDLC.Methods:Immunohistochemistry and immunofluorescence were used to detect the location and expression of HIF-1α in hPDLC cells under different oxygenconcentration.Immunoblotting was used to detect the expression of HIF-1αand IGF-1 proteins in hPDLC cells under different oxygen concentration.Quantitative PCR was used to detect the genes related to osteogenic differentiation and Wnt/β-catenin signaling pathway.Results: With the decrease of oxygen concentration,the expression of HIF increased gradually,and gradually transferred from cytoplasm to nucleus,and the expression of HIF at both the level of mRNA and protein increased with the decrease of oxygen concentration.Real-time fluorescence quantitative PCR data showed that severe hypoxia promoted the expression of osteogenic differentiation genes OPN,ALP,CEMP and CAP.The expression of AXIN2,β-catenin and c-myc,the key genes of Wnt/β-catenin signaling pathway,were detected by Western blotting and quantitative PCR,which indicated that they were highly expressed in hypoxic environment.Conclusion: Hypoxia can promote the osteogenic differentiation of hPDLC cells.Wnt/β-catenin signaling pathway is activated in hypoxia-induced hPDLC.Part 4 Screening of differentially expressed LncRNAs in gingival tissues of periodontitisObjective: To obtain differentially expressed LncRNA in gingival tissues of periodontitis,and to provide theoretical basis for molecular diagnosis of periodontitis and personalized targeted drug therapy.Methods:The expression profiles of LncRNA in gingival tissues of 40 patients withperiodontitis and 20 normal persons were detected by Arraystar LncRNA3.0 chip.The expression of LncRNA in hypoxia-induced hPDLC and periodontitis tissues was detected by Rt-qPCR to verify the results of microarray detection.Results: There were nearly 175 LncRNAs were differentially expressed,with 58 LncRNAs upregulated and 117 LncRNAs downregulated in the periodontitis samples in comparison with the normal periodontal tissues.Subsequently,the following bioinformatic analysis results,including the clustering heat-map analysis,GO and KEGG analysis,showed that the upregulated LncRNAs were involved in the metabolism pathways closely related with hypoxia,while the downregulated LncRNAs were mainly participated in the cellular inflammatory processes.The expression of LncRNA-01126 in periodontitis tissues increased with the decrease of oxygen concentration in hPDLC cells,and was significantly correlated with the expression of HIF-1α.Conclusion: Hypoxic environment may induce the expression of LncRNA-01126 and participate in hypoxic stress response in periodontitis.