The Up-regulatory Effects Of Nicotine To Long Noncoding RNA NEAT1 On Inflammatory Reaction In Human Periodontal Ligament Cells

As the role of conjunction between dental hard tissue and alveolar bone,periodontal ligament is the most important component of periodontal tissue.Periodontitis is the disease,which has seriously affects on oral health.Smoking not only increases the incidence and severity of periodontitis,but reduces the effect of periodontal treatment in both surgical and non-surgical ones as well.It has been found that nicotine produced by tobacco smoke can cause adverse effects on many organs and tissues of the human body,including periodontal tissue.Previous studies have revealed that as a ligand specific binding with acetylcholine receptor in human periodontal ligament cells,nicotine plays a significant role in inflammatory expression in periodontal ligament cells,but its molecular mechanism is still undercover.Moreover,it is shown that lnc RNA is a kind of non-coding RNA,which is widely present in cells,and is involved in the regulation of gene transcription and inflammatory response.However,the reporters is rare that the effect on nicotine to lnc RNA in the inflammatory response.Thus,we hypothesized that after binding with the acetylcholine receptor,nicotine could induce the inflammatory injury of the periodontal tissues by regulate the expression of lnc RNA.However,the specific mechanism still needs to be explored.In this study,we are going to explore the role of lnc RNA in the smoking-related periodontitis,and provide a new approach for the control and cure of smoking-related periodontitis.【Aims】This experiment is aimed to explore the expression of alpha 7 n ACh R and lnc RNA NEAT1 in human periodontal ligament cells by time-and dose-response on nicotine,and revealed the effect of long non-coding RNA NEAT1 on the expression of inflammatory cytokines and the possible mechanisms in the pathogenesis of smoking-related periodontitis.【Materials and methods】1.The human periodontal ligament cells were cultured by the modified tissue block method that combined with enzymatic digestion,and the cell type was identified by flow cytometry analysis.2.The fifth generation of human periodontal ligament cells were given nicotine in different time and dose,and then detected the expression of NEAT1 by real-time quantitative PCR.We used the real-time quantitative PCR and Western Blot to detect of α7 n ACh R.After determining the appropriate time and dose,Human periodontal ligament cells were treated by the stimulation of nicotine and / or α-BTX,and then the cells were detected the expression of NEAT1 by real-time quantitative PCR and fluorescence in situ hybridization.3.The expression of IL-1β,IL-6,IL-8 and TNF-αwas detected by real-time quantitative PCR in fifth generations of human periodontal ligament cells stimulated by nicotine,and then verified by ELISA.Then use si RNA to inhibit the expression of NEAT1 and give the nicotine stimulation to observe the expression of inflammatory cytokines.Finally,use NEAT1 plasmid to amplify the expression of NEAT1 and observe the expression of inflammatory cytokines.Analyze data and explore the relationship between NEAT1 and inflammatory factors.4.Nicotine were given to the fifth generation of human periodontal ligament cells in different time and dose to observe the expression and distribution in the nucleus of SFPQ and NONO by Western Blot and immunofluorescence.After nicotine stimulation and si RNA inhibition of SFPQ or NONO,the expression of inflammatory cytokines was detected by real-time quantitative PCR.【Results】1.Successfully cultured human periodontal ligament cells.The results of flow cytometry analysis showed that the expression of CD105 and CD90 were positive,the expression of CD14 and CD31 was negative.2.Nicotine can increase the expression level of NEAT1 in human periodontal ligament cells in a certain time and dose range.We select 10-5M,2h nicotine stimulation as the observation stimulation on NEAT1 in human periodontal ligament cells.When nicotine increased NEAT1,the expression of 7n ACh R was increased,and the expression of NEAT1 was unchanged when nicotine was inhibited to combine with α7 n ACh R.3.After the expression of NEAT1 was increased by the effect of nicotine in human periodontal ligament cells,only IL-8 expression was increased.The expression of IL-8 was decreased after inhibiting the expression of NEAT1,and the expression of NEAT1 increased with the increase of the expression of IL-8.4.While the expression of protein SFPQ and NONO in nucleus did not change,the state of distribution changed from dispersion to aggregation after nicotine stimulated human periodontal ligament cells.Comparing with nicotine stimulation,the expression of IL-8 increased more after inhibiting the expression of SFPQ,while there was a not similar change observed in inhibiting NONO group.【Conclusions】Human periodontal ligament cells were successfully cultured and identified.Nicotine can up-regulate the expression of α7 n ACh R and NEAT1 in human periodontal ligament cells,and the up-regulating expression of NEAT1 was caused by α7 n ACh R.In human periodontal ligament cells,nicotine increased the expression of IL-8 by up-regulating the expression of NEAT1,and the increase was related to the nucleus protein SFPQ.