Study On The Molecular Mechanism Of MiR-146a And MiR-155-5p In Rheumatoid Arthritis

Objective: The main purpose of this study was to clarify the role of miR-146 a and miR-155-5p in the pathogenesis of rheumatoid arthritis(RA),and to explore the molecular mechanism of its action,so as to provide a theoretical reference for the study of the pathological mechanism of RA and other inflammatory diseases.Methods: Construction of inflammatory functional cell model: Three types of cells closely related to RA inflammatory response: human umbilical vein endothelial cells(HUVECs),human peripheral blood mononuclear cells(PBMCs)and rheumatoid arthritis synovial fibroblasts(HFLS-RA)were stimulated by LPS or transiently transfected with mir-146 a and mir-155-5p simulants to establish relevant cell models.Relative quantitative real-time fluorescence quantitative polymerase chain reaction(real-time PCR)was used to detect the dynamic expression of two inflammatory target genes(TLR4 and NF-kappa B)in target cells.Stem ring primer-RT-q PCR was used to detect the dynamic expression of microRNA-146 A and microRNA-155-5p in target cells.Then SPSS17.0 software was used to conduct t test and one-way analysis of variance on the data,so as to study the role of miR-146 a and miR-155-5p in the inflammatory response,and to elucidate the specific regulatory mechanism of the inflammatory markers TLR4 and NF-kappa B.Results: Part ?: Construction of HUVECs inflammatory cell model and Study on the expression of target microRNA: HUVECs were stimulated with 0.1,1,10 g/mL LPS and the expression changes of each gene at 0,4,8,12,24 and 48 h after LPS stimulation were detected.The expressions of miR-146 a,miR-155-5p,TLR4 and NF-kappa B were all increased.The expression of TLR4 and NF-kappa B showed LPS concentration dependence.And the up-regulation of miR-155-5p is preferred over miR-146 a,TLR4 and NF-kappa B.Part ?: Establishment of case-control PBMCs model and Study on the expression of target microRNA: PBMCs were isolated from blood samples of RA patients and healthy volunteers(HC).The expression of target genes was detected directly.Compared with HC,miR-146 a,miR-155-5p,and TLR4 were up-regulated in RA patients(p < 0.05),while the expressions of NF-kappa B were not significantly different(p > 0.05).The optimized cultured PBMCs were stimulated with 0.1 g/mL LPS.After the stimulation of 0,24,48,72 h or 96 h,miR-155-5p,TLR4 and NF-kappa B in HC PBMCs were up-regulated at different time periods(p < 0.05),while the expression of miR-146 a was basically unchanged(p > 0.05).In the PBMCs of RA,TLR4 was significantly down-regulated(p < 0.01),and miR-146 a was significantly upregulated to the peak at 24 h(p < 0.05),and then returned to the original level.While the expression of miR-155-5p and NF-kappa B showed no significant difference(p > 0.05).The third part: Construction of overexpression models of miR-146 a,miR-155-5p in HUVECs and HFLS-RA Cells and Study on Regulation of miR-146 a,miR-155-5p: HUVECs and HFLS-RA cells were transfected instantaneously with miR-146 a and miR-155-5p mimics.When HUVECs were transfected with miR-146 a mimic,compared with control group,at 24 hours,miR-155-5p was up-regulated,while TLR4 and NF-kappa B remained unchanged;at 48 hours,the levels of miR-155-5p and TLR4 were significantly increased(p < 0.05),while the levels of NF-kappa B remained unchanged(p > 0.05).When HUVECs were transfected with miR-155-5p mimic,the expression of miR-146 a,TLR4 and NF-kappa B increased significantly at 24 and 48 hours(p < 0.05).In HFLS-RA cells,the expression of miR-155-5p,TLR4 and NFkappa B remained unchanged at 24 h after transfection with miR-146 a mimic.At 48 h,the expression of miR-155-5p and TLR4 increased significantly(p < 0.05),while the expression of NF-kappa B had no significant difference.The expression of miR-146 a,TLR4 and NF-?B was significantly increased at 24 h and 48 h after transfection of HUVECs with miR-155-5p mimics(p<0.05).Conclusion: Through the experiments in this paper,the following conclusions can be drawn: 1.miR-146 a and miR-155-5p responded to LPS-induced inflammatory stimulation.2.The expression of miR-146 a and miR-155-5p is closely related to the type and state of cells.3.The regulation of inflammatory response by miR-146 a and miR-155-5p is related to the inflammatory state of cells.In the process of normal inflammation,the expression of NF-kappa B can be regulated synergistically by miR-146 a and miR-155-5p,so as to maintain inflammation homeostasis.However,the negative regulation of miR-146 a is dose-and time-dependent.In uncontrolled inflammatory response,the pro-inflammatory effect of miR-155-5p was dominant,and it antagonized the negative regulation of miR-146 a on inflammatory response.