Regulation Of MiRNA-126 On The Proliferation Of Macrophages Stimulated By High Glucose

Objective:In recent years,more and more studies had found that there were two-way effects between diabetes and periodontitis through inflammatory response,in which microRNAs(miRs)played an important role.Macrophages,as immune cells,played a critical role in phagocytosis,inflammation and tissue remodeling.The study was to explore the effects of high glucose on the proliferation of THP-1-derived macrophages,and the functions of miR-126 on the proliferation of THP-1-derived macrophages in high glucose environment,and to preliminarily declare the relationship between diabetes and periodontitis.Methods:1.When THP-1 cells grew well in vitro,phorbol-12-myristate-13-acetate(PMA)was added in culture medium at a final concentration of 5ng/ml.After incubation for 48 h,observed the morphological changes of the cells and confirmed they had differentiated into macrophages.2.THP-1-derived macrophages were cultured with low glucose(5.5mmol/l),medium glucose(15mmol/l),and high glucose(25mmol/l)for 1,3,5 and 7 days.Cell counting kit-8(CCK-8)method was used to detect the proliferation of THP-1-derived macrophages and calculate the cell proliferation activity.3.After cells were cultured with different glucose concentration for 1,3,5 and 7 days,real time polymerase chain reaction(real-time PCR)detected the expressions of miR-126,caspase-3,Bcl 2-associated X protein(BAX),B-cell lymphoma-2(Bcl-2),phosphoinositol-3 kinase regulatory subunit 2(PIK3R2),transforming growth factor-beta 3(TGF-β3).4.THP-1-derived macrophages were cultured in low,medium and high glucose media,and transfected with miR-126 mimic,inhibitor or scramble RNA.The transfection efficiency was detected by confocal laser scanning microscope and real-time PCR.5.After cell transfection for 72 h,CCK-8 method was used to detect the proliferation of THP-1-derived macrophages.6.After cell transfection for 72 h,the levels of miR-126,caspase-3,BAX,Bcl-2,PIK3R2,TGF-β3 were detected by real-time PCR.Result:1.THP-1 cells could be induced into macrophages effectively with 5ng/ml PMA.2.With the concentration of glucose increase,the proliferation of THP-1-derived macrophages was inhibited,and the survival rate of macrophages decreased.3.Increasing glucose concentration promoted the expression of miR-126,BAX,caspase-3 and TGF-β3 significantly(P<0.05),and suppressed Bcl-2,PIK3R2 expression(P<0.05).4.After transfection of miR-126 mimic,the proliferation ability of THP-1-derived macrophages decreased under different glucose concentrations.The level of miR-126 significantly increased(P<0.05);both m RNA levels of BAX,caspase-3 and TGF-β3 significantly increased(P<0.05),but Bcl-2 and PIK3R2 decreased(P<0.05).5.After transfection of miR-126 inhibitor,the proliferation ability of macrophages increased.The level of BAX,caspase-3 and TGF-β3 significantly decreased(P<0.05),but Bcl-2 and PIK3R2 increased(P<0.05).Conclusion:1.High glucose could inhibit the proliferation of THP-1-derived macrophages and increase the expression of miR-126.2.MiR-126 could inhibit the proliferation of THP-1-derived macrophages in high glucose environment by up-regulating the expression of BAX,caspase-3 and TGF-β3 and down-regulating the expression of Bcl-2 and PIK3R2.However,the specific mechanism needs to be explored and verified in the future.