| Objective:To investigate whether miR-223-3p can be involved in regulating the inflammatory response in preeclampsia(PE)placenta by targeting NLRP3 and the possible mechanisms of action.Methods:1.60 pregnant women with cesarean section in Subei people’s Hospital of Jiangsu Province from December 2018 to January 2020 were selected,including 30 cases in the PE group and 30 cases in the normal control group.Real-time quantitative polymerase chain reaction(RT-qPCR),western blot(WB)and immunohistochemistry(IHC)were used to evaluate the expression patterns of NLRP3 and miR-223-3p in placenta tissues.2.The potential binding sites of miR-223-3p and NLRP3 3’Untranslated Region(UTR)were predicted by bioinformatics software.Then the targeting relationship between miR-223-3p and NLRP3 was explored through dual-luciferase reporter gene assay by constructing miR-223-3p and NLRP3 3 ’UTR-wild type(wt)/mutant type(mut)luciferase reporter vectors,respectively.3.Different concentrations of lipopolysaccharide(LPS)(0,50,100,200,500,1000 ng/mL)were used to induce HTR8/SVneo cells for 24 h.RT-qPCR and WB were used to detect the expression levels of NLRP3 inflammasome components NLRP3 and Caspase-1,pyrocytosis related protein GSDMD at mRNA and protein levels,respectively.And the secretion levels of Interleukin 1β(IL-1β)and IL-18 were detected by enzyme-linked immunosorbent assay(ELISA).These were used to determine the optimal conditions for constructing a PE placental trophoblast inflammation model.4.miR-223-3p overexpression or miR-223-3p negative control plasmids were transfected into the PE inflammatory model.The regulatory effects of miR-223-3p overexpression on the expression levels of NLRP3 inflammasome components(NLRP3,caspase-1),pyrocytosis related protein(GSDMD)expression and the secretion levels of downstream inflammatory factors(EL-1β,IL-18)were evaluated by RT-qPCR,WB and ELISA.Results:1.Compared with the normal controls,the expression of NLRP3 in PE placenta was significantly upregulated at mRNA and protein levels(P<0.05),and the NLRP3 protein was mainly expressed in syncytiotrophoblasts,stromal cells and endothelial cells of the placenta.However,the expression of miR-223-3p was significantly downregulated in PE placenta(P<0.05).2.Dual-luciferase reporter gene assay showed that miR-223-3p overexpression plasmids could specifically bind to NLRP3-3’UTR-wt plasmids in HEK-293T cells,significantly reducing the luciferase activity of cells(P<0.001);while the miR-223-3p overexpression plasmid could not interact with NLRP3-3’UTR-mut,and there was no significant change in the luciferase activity of cells.These results indicated that NLRP3 was a direct target gene of miR-223-3p.3.In vitro cell experiments,the expressions of NLRP3,Caspase-1,and GSDMD in HTR8/SVneo cells at the mRNA and protein levels gradually increased with the increase of LPS concentration.Especially when LPS was 500 ng/mL,the expression level of the above genes were most significantly increased(P<0.001).The secretion levels of inflammatory factors IL-1β and IL-18 were also in a LPS concentration-dependent manner,and the increase was most obvious when LPS was 500 ng/mL(P<0.001).It was concluded that 500 ng/mL LPS stimulated HTR8/SVneo cells for 24 h was the best condition to construct PE cell inflammation model.4.In the inflammatory model,miR-223-3p overexpression could effectively inhibit the expression levels of NLRP3 inflammasome components(NLRP3,Caspase-1)and pyrocytosis related protein(GSDMD)(P<0.05),as well as the secretion levels of downstream inflammatory cytokines(IL-1β and IL-18)(P<0.05).Conclusion:miR-223-3p may suppress the activation of NLRP3 inflammasome,the pyroptosis of trophoblast cells,as well as the secretion of downstream inflammatory factors in PE placenta by targeting NLRP3,providing a new potential treatment strategy for PE. |
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