| Objective:To explore the role of miR-22-3p and its target protein NLRP3 signaling pathway in human microvascular endothelium cell(HBMEC)after escherichia coli infection in the process of pyroptosis and its related mechanisms.Methods:(1)HBMEC were cultured in vitro and randomly divided into 4 groups.Except the control group,which was treated with phosphate buffer salt solution(PBS),the other 3 groups were respectively treated with E.coli K1 for 1h,2h and 3h.The expression of miR-22-3p and IL-1β,IL-18,IL-10 and TNF-α m RNA in each group were detected by q RT-PCR.Western blot analysis of NLRP3,GSDMD-F,GSDMD-N,pro-caspase-1 and cleaved caspase-1 protein levels in all groups.The expression of VE-Cadherin was detected after 3h by immunofluorescence.(2)NLRP3 wild-type and mutant vectors were respectively constructed and co-transfected into 293 T cells with miR-22-3p mimics.Luciferase assay was performed to detect luciferase activity.(3)HBMEC models with overexpression and underexpression of miR-22-3p were constructed and randomly divided into four groups: NC mimics group,miR-22-3p mimics group,NC inhibitor group and miR-22-3p inhibitors group.The transfection success rate was detected by q RT-PCR.Western blot was used to detect the protein levels of NLRP3 in each group.(4)NLRP3 si RNA was constructed in vitro and transfected into HBMEC.The cells were divided into four groups: si NC group,si NLRP3 group,E.coli+si NC group,and E.coli+si NLRP3 group.The m RNA expressions of IL-1β,IL-18,IL-10 and TNF-α in each group were detected by q RT-PCR.Western blot analysis of NLRP3,GSDMD-F,GSDMD-N,pro-caspase-1 and cleaved caspase-1 protein levels.(5)HBMEC were cultured in vitro and randomly divided into 4groups: NC mimics group,miR-22-3p mimics group,E.coli+NC mimics group,and E.coli+ miR-22-3p mimics group.The m RNA expressions of IL-1β,IL-18,IL-10 and TNF-α in each group were detected by q RT-PCR.Western blot analysis of NLRP3,GSDMD-F,GSDMD-N,pro-caspase-1 and cleaved caspase-1 protein levels.(6)HBMEC were cultured in vitro and randomly divided into 5groups.NC mimics+si NC group,E.coli+NC mimics+si NC group,E.coli+miR-22-3p mimics+si-NC group and E.coli+miR-22-3p mimics+si NLRP3 group.Western blot was used to detect GSDMD-F,GSDMD-N,pro-caspase-1 and cleaved caspase-1 protein levels in cells at each time point.The expression of IL-1β,IL-18,IL-10 and TNF-α m RNA were detected by q RT-PCR.(7)HBMEC were cultured in vitro and randomly divided into 7groups: si NC group,si NLRP3 group,E.coli+si NLRP3 group,NC mimics,miR-22-3p mimics group,E.coli+ miR-22-3p mimics group and E.coli+miR-22-3p mimics group + si NLRP3 group,the expression of VE-cadherin was detected by immunofluorescence.Results:(1)The expression of miR-22-3p was significantly downregulated at each time point after E.coli infection compared with the control group(P?0.001),and the expression level was the lowest at 3h after infection.However,GSDMD-F,GSDMD-N,pro-caspase-1,cleaved caspase-1 protein levels and IL-1β,IL-18,TNF-α m RNA levels were significantly higher than those in the control group(P?0.001),and the expression levels were the highest at 3h.The expression of IL-10 m RNA showed an opposite trend.(2)The luciferase activity was significantly decreased in the NLRP3wild-type vector transfection group,but not significantly changed in the NLRP3 mutant vector transfection group,and confirmed that NLRP3 is the target gene of miR-22-3p.(3)Overexpression of miR-22-3p significantly decreased NLRP3 m RNA and protein levels(P?0.001),while inhibition of miR-22-3p significantly upregulated NLRP3 m RNA and protein levels in HBMEC(P?0.001).(4)NLRP3 silencing significantly inhibited pro-caspase-1,cleaved caspase-1,GSDMD-F,GSDMD-N protein levels and IL-1β,IL-18,and TNF-α m RNA levels in HBMEC induced by E.coli infection.Increased IL-10 m RNA expression(P?0.001).(5)MiR-22-3p overexpression significantly down-regulated the levels of NLRP3,pro-caspase-1,cleaved caspase-1,GSDMD-F,and GSDMD-N proteins,and decreased the m RNA levels of NLRP3,IL-1β,IL-18,and TNF-α.Increased IL-10 m RNA expression(P?0.001).However,The inhibitory effect on cell pyroptosis was significantly weakened when NLRP3 was silenced and miR-22-3p was overexpressed at the same time.The levels of GSDMD-F and GSDMD-N were also significantly upregulated(P?0.001).(6)Immunofluorescence results showed that the expression of VE-cadherin decreased significantly in HBMEC after E.coli infection,and NLRP3 silencing and miR-22-3p overexpression can up-regulated the expression of VE-cadherin respectively in cells after E.coli infection.After co-transfection with si NLRP3 and miR-22-3p mimics,the expression of VE-cadherin in E.coli+si NLRP3 or E.coli+ miR-22-3p mimics was significantly down-regulated(P < 0.05).Conclusion:(1)E.coli induced HBMEC pyroptosis and endothelial cell adhesion injury,reduced the expression of miR-22-3p in HBMEC,and activated NLRP3 inflammasome and inflammatory responses.(2)NLRP3 is the target gene of miR-22-3p.(3)MiR-22-3p overexpression can inhibit E.coli induced HBMEC pyroptosis and endothelial cell adhesion injury,and the mechanism may be related to the negative regulation of NLRP3 mediated caspase-1/GSDMD pyroptosis signaling pathway and the release of inflammatory mediators.There are 12 figures,9 tables and 81 references... |
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