The Role And Regulation Mechanism Of Long Non-coding RNA MALAT1 In Chronic Periodontitis

Objective:The purpose of this study was to detect the differential expression of metastasis-associated lung adenocarcinoma transcript 1(MALAT1)in chronic periodontitis;to find out the candidate target gene of MALAT1 by bioinformatics;to explore the regulatory pathway of MALAT1 on inflammatory cytokines in human gingival fibroblasts(HGFs);to clarify the function and mechanism of MALAT1 in chronic periodontitis;and to provide experimental evidence for the treatment of chronic periodontitis.Methods:1.After informed consent obtained,gingival tissues of healthy volunteers and chronic periodontitis patients were collected.Real-time polymerase chain reaction was used to detect the expression level of MALAT1.2.Bioinformatics analysis found that MALAT1 and toll-like receptor 4(TLR4)might bind with micro RNA(mi R)-20 a.Dual luciferase reporter assays and RNA binding protein immunoprecipitation(RIP)experiments were used to verify and detect the binding sites.3.After informed consent obtained,gingival tissues were collected during extracting the impacted third molars.HGFs were cultured in vitro by tissue explants method.Different concentrations(0,0.1,1,10?g/ml)of Porphyromonas gingivalis(P.g)lipopolysaccharide(LPS)or Escherichia coli(E.coli)LPS were used to stimulate HGFs.And the expression levels of MALAT1,mi R-20 a,TLR4,IL-1?,IL-6,IL-8 and TNF-? were detected by real time PCR at 6 h,12 h and 24 h after stimulation.4.After transfection with MALAT1 si RNA or mi R-20 a mimics,the transfection efficiency was detected.After transfection,HGFs were stimulated with 1?g/ml P.g LPS or E.coli LPS.Then,expression levels of MALAT1,mi R-20 a,TLR4,IL-1?,IL-6,IL-8 and TNF-? were detected.5.Over-expression plasmid of MALAT1 was constructed and the transfection efficiency was determined.After transfection,HGFs were stimulated with 1?g/mlP.g LPS or E.coli LPS.Then,expression levels of MALAT1,mi R-20 a,TLR4 and proinflammatory cytokines were detected.Results:1.Compared with healthy gingival tissues,the expression of MALAT1 in chronic periodontitis was significantly increased.2.The dual luciferase reporter assay demonstrated that MALAT1 and TLR4 bound with mi R-20 a.Furthermore,RIP experiments demonstrated that MALAT1,mi R-20 a and TLR4 were located in the RNA binding protein complex formed by Ago2.3.The expression levels of MALAT1,TLR4,IL-1?,IL-6,IL-8 and TNF-?increased while the expression of mi R-20 a decreased after E.coli LPS or P.g LPS stimulation at different concentrations.4.After transfection of HGFs with MALAT1 si RNA,the expression of MALAT1 significantly decreased(P<0.05),and the level of mi R-20 a significantly increased while TLR4,IL-1?,IL-6,IL-8 and TNF-? significantly decreased(P<0.05)whatever the cells were stimulated with LPS.5.After transfection of HGFs with mi R-20 a mimics,the expression of mi R-20 a significantly increased(P<0.05)and the expression levels of MALAT1,TLR4,IL-1?,IL-6,IL-8 and TNF-? significantly decreased(P<0.05)whatever the cells were stimulated with LPS.6.After transfection of HGFs with p ZW1-sno Vector-MALAT1,the expression of MALAT1 significantly increased(P<0.05).And the expression levels of mi R-20 a significantly decreased while TLR4,IL-1?,IL-6,IL-8 and TNF-? significantly increased(P<0.05)whatever the cells were stimulated with LPS.Conclusion:1.MALAT1 is up-regulated in inflammatory gingival tissues of chronic periodontitis,and promotes the expressions of inflammatory cytokines IL-1?,IL-6,IL-8 and TNF-?,suggesting that it is involved in the inflammatory process of chronic periodontitis.2.The interaction between MALAT1 and mi R-20 a regulates the expression of TLR4.3.Mi R-20 a inhibits the expression of TLR4 and proinflammatory cytokines via binding to TLR4 3'-UTR.4.MALAT1-mi R-20a-TLR4 signaling regulates the expressions of inflammatory cytokines,which is expected to become a novel therapeutic target for chronic periodontitis.