Changes in the ability to clear Mycobacterium tuberculosis,apoptosis,inflammatory response of mac-rophages transfected with miR-223 mimics and the targeted relationship between miR-223 and NLRP3
Objective To observe the changes in serum microRNA-223(miR-223)levels in patients with pulmo-nary tuberculosis,as well as the changes in the clearance ability to clear Mycobacterium tuberculosis(MTB)H37Rv,apoptosis and inflammatory response of macrophages transfected with miR-223 mimics,and to analyze the targeted relation-ship between miR-223 and NLRP3.Methods Thirty-seven serum samples from active pulmonary tuberculosis patients(tuberculosis group)and 40 serum samples from healthy individuals during the same period(healthy group)were select-ed,and miR-223 was detected in the serum samples of both groups using RT-PCR.Human monocyte line THP-1 was cul-tured in vitro and differentiated into macrophages after 24 h of stimulation with phorbol.Macrophages were randomly divid-ed into the healthy group,MTB group,MTB+mimics-NC group,and MTB+miR-223 mimics group.Cells in the healthy group were cultured routinely,while cells in the MTB group,MTB+mimics-NC group,and MTB+miR-223 mimics group were all infected with MTB standard strain H37Rv.After infection,in the MTB+mimics-NC group and MTB+miR-223 mimics group,we used liposome transfection method to transfect mimics-NC and miR-223 mimics into cells,respective-ly.The miR-223,NLRP3,TNF-α and IFN-γ mRNA in cells of each group were detected by RT-PCR.Flow cytometry was used to detect the apoptosis rate of cells in each group,and colony forming unit(CFU)counting method was used to detect the H37Rv bacterial load in each group.THP-1 cells in the logarithmic growth phase were taken and randomly di-vided into the miR-223 mimics+NLRP3-WT group,the mimics-NC+NLRP3-WT group,the miR-223 mimics+NLRP3-MUT group,and the mimics-NC+NLRP3-MUT group,respectively.The miR-223 mimics and NLRP3-WT,mimics-NC and NLRP3-WT,miR-223 mimics and NLRP3-MUT,and mimics-NC and NLRP3-MUT were co-transfected into cells of the above groups,respectively,by Lipofectamine 2000 reagent in accordance with the operation instructions of the kit.The relative luciferase activity of cells in each group was detected at 24 h after transfection.The dual luciferase reporter gene assay was used to verify whether there was a targeted relationship between miR-223 and NLRP3.Results Com-pared with the healthy group,the serum miR-223 expression decreased in the tuberculosis group(P<0.05).Compared with the healthy group,the miR-223 expression and apoptosis rate decreased,and the NLRP3,TNF-α,IFN-γ mRNA and bacterial load increased in the MTB group(all P<0.05).Compared with the MTB+mimics NC group,the MTB+miR-223 mimics group showed increased miR-223 expression and cell apoptosis rate,and decreased expression levels of NL-RP3,TNF-α,IFN-γ mRNA and bacterial load(all P<0.05).The dual luciferase reporter gene experiment showed that NLRP3 was a targeted gene for miR-223.Conclusions The expression of serum miR-223 decreases in patients with pulmonary tuberculosis.Transfection of miR-223 mimics can promote macrophage apoptosis,enhance the bactericidal ability of macrophages infected with MTB,and alleviate inflammatory response.The mechanism may be related to target-ed regulation of NLRP3.
microRNA-223Mycobacterium tuberculosismacrophagespulmonary tuberculosisactive pulmo-nary tuberculosisNOD-like receptor heat protein domain-related protein 3
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