Mechanism Of LncRNA IGHCγ1/miR-6891-3p/TLR4 CeRNA Network Regulating Macrophage-mediated Immune And Inflammatory Response In Osteoarthritis

Objective:LncRNA IGHCγ1 has been demonstrated to exert the inflammatory and immunomodulatory effects,whereas the specific role and mechanism of lnc RNA IGHCγ1 in osteoarthritis(OA)remains not clear at home and abroad.The present study is aimed to elucidate the mechanism and clinical significance of lnc RNA IGHCγ1 in regulating macrophage inflammatory response via targeting mi R6891-3p/TLR4 in OA.Methods:Eighty-eight OA patients and 36 healthy controls from the Affiliated Hospital of Qingdao University and Rizhao People’s Hospital of Shandong Province were enrolled in this study.1 Density gradient centrifugation: isolating the peripheral blood mononuclear cells(PBMCs).2 Construction of macrophage lines by intervening the expression of lncRNA IGHCγ1 and TLR4: to stimulate and induce THP-1 cells to differentiate into macrophage-like cells(p THP-1 cells)using PMA and construct lnc RNA IGHCγ1 or TLR4-up-or-downregulated pTHP-1 macrophages using PcDNA3.1 lentiviral vectors.3 CCK-8 and Ed U experiments: evaluating the proliferation of macrophages.4 FISH assay: to define the expression and localization of lncRNA IGHCγ1 in pTHP-1cells.5 Real-time PCR: to detect the m RNA levels of IL-6,TNF-α,TLR4 and lncRNA IGHCγ1in PBMCs and p THP-1 cells.6 ELISA: to test the protein expression levels of IL-6 and TNF-α in the cell culture supernatant of mi R-6891-3p mock-like cells treated with or without p THP-1 cells for 24 hours.7 Western blot: to detect the protein expression level of TLR4 and p-NF-κB.8 Luciferase reporter assay: 293 T cells were transfected with mi R-6891-3p inhibitors,mimics,and/or Lv-TLR4-WT,Lv-L-TLR4-MT and/or Lv-IGHCγ1-WT and Lv-IGHCγ1-MT reporter plasmids.The luciferase activity was determined.9 ELISA: to estimate changes of NF-κB activity.10 RNA-binding protein immunoprecipitation assay(RIP): Co-immunoprecipitation using Ago2 antibody or Ig G,in which the expression of RNA in the immune complexes was detected by Real-time PCR.Results:1.The expression of LncRNA IGHCγ1 was significantly increased in both PBMCs from OA patients and p THP-1 macrophages.2.Overexpression of lncRNA IGHCγ1 could promote macrophage proliferation,while interfering expression of lnc RNA IGHCγ1 could inhibit macrophage proliferation.3.LncRNA IGHCγ1 was mainly localized in the cytoplasm of macrophages,which might act as a ce RNA molecule for mi R-6891-3p.4.The expression of mi R-6891-3p was significantly reduced in the PBMCs from OA patients,which could inhibit the proliferation of macrophages.5.TLR4 was the targeted m RNA of mi R-6891-3p,which promoted macrophage proliferation and the production of inflammatory cytokines IL-6 and TNF-α.6.LncRNA IGHCγ1 promoted macrophages-mediated inflammatory response through the mi R-6891-3p/TLR4 ce RNA network.7 The ceRNA regulation of lncRNA IGHCγ1 on mi R-6891-3p/TLR4 in macrophages was dependent on the activation of the NF-κB signaling pathway.Conclusions:1.LncRNA IGHCγ1 is upregulated in PBMCs from OA patients,whereas mi R-6891-3p is downregulated.They are involved in regulating macrophage-mediated biological effects in OA.2.LncRNA IGHCγ1 targets mi R-6891-3p/TLR4 through the ceRNA mechanism and enhances macrophage-mediated immune and inflammatory response in OA by activating NF-κB signaling pathway.