A Preliminary Study On The Role And Regulation Mechanism Of LncRNA-TNFRSF13C In Aggressive Periodontitis

Objective: In our previous study,long non-coding RNA TNFRSF13 C significantly increased in gingival tissues of aggressive periodontitis compared with normal gingival tissues,which suggest that long non-coding RNA TNFRSF13 C may be involved in the process of aggressive periodontitis.This study was to detect the expression of lncRNA-TNFRSF13 C,Jun,SP1,TCF20,MMP-1 and MMP-3 in human gingival tissues under different inflammatory conditions.To detect the regulation of lncRNA-TNFRSF13 C on Jun,SP1,TCF20 a nd MMP-1,MMP-3 by in vitro cell assay.This study is to explore the role and regulation mechanism of lncRNA-TNFRSF13 C in aggressive periodontitis,and to provide a new theoretical basis for elucidating the pathogenesis of aggressive periodontitis.Methods: ? After informed consent,the gingival tissues of patients with periodontal healthy and aggressive periodontitis were collected,total RNA was extracted by Tri Zol method,and real time polymerase chain reaction was performed.The expression of lncRNA-TNFRSF13 C,Jun,SP1,TCF20,MMP-1 and MMP-3 in gingival tissues from different inflammatory states were examined.2.After the patient's informed consent,the fresh healthy gingival tissue was collected from the healthy volunteers in the department of Oral and Maxillofacial Sur gery,Stomatological Hospital of Tianjin Medical University when they extracted the impacted teeth.HGFs were cultured by tissue block adherence method.The cells at 3-4th generation were obtained,then stimulated with lipopolysaccharide(LPS)of Escherichia coli(E.coli)or porphyromonas gingivalis(P.g)at different concentrations(0?g/ml,0.1?g/ml,1?g/ml,10?g/ml).The expression levels of lncRNA-TNFRSF13 C,Jun,SP1,TCF20,MMP-1 and MMP-3 were detected by real time PCR after 1d?3d?5d and 7d in vitro.3.LncRNA-TNFRSF13 C overexpression plasmid was constructed.After transfection with the plasmid,transfection efficiency into cell was measured.The expression changes in mRNA and protein secretion levels of the above factors were detected after plasmid transfection.4.The lncRNA-TNFRSF13 C siRNA was transfected into HGFs to detect the cell transfection efficiency.The expression changes in mRNA and protein secretion levels of the above factors were detected after siRNA transfection.Results: 1.When comparing with healthy gingival tissues,the expression level of lncRNA-TNFRSF13 C and Jun,SP1,TCF20,MMP-1 and MMP-3 in gingival tissues of patients with aggressive periodontitis was significantly increased.2.After stimulation with different concentrations and time of P.g LPS or E.coli LPS,the expression of lncRNA-TNFRSF13 C and Jun,SP1,TCF20,MMP-1 and MMP-3 increased.3.After transfection of HGFs with Sno-TNFRSF13 C,the expression of lncRNA-TNFRSF13 C significantly increased,compared with the blank vector group(p<0.05);in addition,the expressions of J un,SP1,TCF20,MMP-1 and MMP-3 in sno-TNFRSF13 C group significantly increased(p<0.05).4.After transfection of HGFs with lncRNA-TNFRSF13 C siRNA,the expression of lncRNA-TNFRSF13 C was significantly lower than that in the negative control group(p<0.05).In addition,the expressions of Jun,SP1,TCF20,MMP-1 and MMP-3 in si-TNFRSF13 C group significantly decreased(p<0.05).5.The supernatant of HGFs respectively transfected with si-TNFRSF13 C and sno-TNFSRF13 C was detected by ELISA.The protein levels of MMP-1 and MMP-3 were significantly lower in the si-TNFRSF13 C group compared with the negtive control group(p<0.05).Compared with the blank vector group,MMP-1 and MMP-3 protein levels in sno-TNFRSF13 C group significantly increased(p<0.05).Conclusion: 1.The high expressions of lncRNA-TNFRSF13 C,Jun,SP1,TCF20 and MMP-1 and MMP-3 in inflammatory gingival tissues suggest that these factors are involved in the pathogenesis of aggressive periodontitis.2.lncRNA-TNFRSF13 C can regulate the activity of Jun,SP1 and TCF20,as well as the expression levels of MMP-1 and MMP-3,but the regulatory mechanism needs further exploration.