| Part Ⅰ Pyroptosis is involved in hypoxia/reoxygenation injury of H9C2 cellsObjectives:To investigate whether pyroptosis is involved in hypoxia/reoxygenation injury of H9C2 cells.Methods:H9C2 cells were used to establish hypoxia/reoxygenation injury model and we set different time gradients.The expression of caspase-1 and GSDMD were detected by Western Blot,and the expression of IL-18 and IL-1β were determined by qPCR test.Results:In the hypoxia/reoxygenation injury model of H9C2 cells,after gradually elevated to the peak level during the first 6 hours after reoxygenation,the expression of caspase-1,GSDMD,IL-18 and IL-1β decreased in the following hours.The pyroptosis related proteins and molecules at the peak were significantly higher in hypoxia/reoxygenation injury group than those in the control group(P all<0.05).Conclusions:The pyroptosis is involved in hypoxia/reoxygenation injury of H9C2 cells.Part Ⅱ LncRNAKCNQ1OT1 mediates pyroptosis in H9C2 cells with hypoxia/reoxygenation injury.Objectives:To investigate whether the expression of pyroptosis related proteins were influenced by LncRNA KCNQ1OT1.Methods:H9C2 cells were divided into 4 groups:control group,hypoxia/reoxygenation injury group(H/R),negative control+hypoxia/reoxygenation injury group(NC+H/R),small interfering RNA against LncRNA KCNQ10T1+hypoxia/reoxygenation injury group(si-KCNQ1OT1+H/R).Cell counting kit-8 was used to detect the cell survival rate.Immunofluorescence staining was used to detect the expression of NLRP3 and IL-1β.qPCR was employed to detect the expression of LncRNA KCNQ1OT1 and miR-214-3p.qPCR and western blot were used to detect the expression of NLRP3,caspase-1,GSDMD,IL-18 and IL-1 p.Results:1.The expression of LncRNA KCNQ1OT1 was increased in H9C2 cells with hypoxia/reoxygenation injury as compared with control group(P<0.05).2.The transfection efficacy and cell survival rate of H9C2 cells transfected with small interfering RNA against LncRNA KCNQ1OT1(si-KCNQ1OT1)were higher than 70%.The expression of LncRNA KCNQ1OT1 in H9C2 cells was successfully inhibited after si-RNA transfection.The expression of miR-214-3p was increased in si-RNA transfected H9C2 cells(P<0.05).3.As compared with NC+H/R group,the cell survival rate was higher in H/R+si-KCNQ1OT1 group(P<0.05).4.The expression of NLRP3 and IL-1β were overexpressed in the H/R group compared with the control group,whereas they were downregulated after the expression of LncRNA KCNQ1OT1 was inhibited by si-KCNQ1OT1(P<0.05).5.The mRNA and protein expression of NLRP3,caspase-1,GSDMD,IL-18 and IL-1βwere significantly downregulated after the expression of LncRNA KCNQ1OT1 was successfully inhibited by si-KCNQ1OT1(P<0.05).Conclusions:The expression of LncRNA KCNQ1OT1 is negatively related to the expression of miR-214-3p,and LncRNA KCNQ1OT1 is associated with pyroptosis in H9C2 cells with hypoxia/reoxygenation injury.Part Ⅲ miR-214-3p mediates pyroptosis in H9C2 cells with hypoxia/reoxygenation injury.Objectives:To investigate whether miR-214-3p could mediate pyroptosis in H9C2 cells with hypoxia/reoxygenation injury.Methods:H9C2 cells were divided into 5 groups:control group,hypoxia/reoxygenation injury group(H/R),negative control+hypoxia/reoxygenation injury group(NC+H/R),down-regulated expression of miR-214-3p+hypoxia/reoxygenation injury group(AMO-214-3P+H/R)and up-regulated expression of miR-214-3p+hypoxia/reoxygenation injury group(miR-214-3p mimics+H/R).Cell counting kit-8 was used to detect the cell survival rate.qPCR was employed to detect the expression of LncRNAKCNQ1OT1 and miR-214-3p and the expression of NLRP3,caspase-1,GSDMD,IL-18 and IL-1β.Results:1.The expression of miR-214-3p was reduced in H9C2 cells with hypoxia/reoxygenation injury(P<0.05).2.The H9C2 cells were transfected with anti-miRNA oligonucleotide of miR-214-3p(AMO-214-3P)and miR-214-3p mimics using lipofectamine(?)2000 reagent.The transfection efficacy and cell survival rate in both AMO-214-3P+H/R and miR-214-3p mimics+H/R groups were higher than 70%.The expression of miR-214-3p in H9C2 cells was successfully inhibited after transfection with AMO-214-3P,and the expression of LncRNAKCNQ1OT1 was markedly increased.In contrast,after transfection with miR-214-3p mimics,the expression of miR-214-3p was significantly increased whereas the expression of LncRNA KCNQ1OT1 was substantially decreased.3.The cell survival rate was positively correlated with the expression of miR-214-3P(P<0.05).4.In H9C2 cells hypoxia/reoxygenation injury model,after transfection with miR-214-3p mimics,the expression of NLRP3,caspase-1,GSDMD,IL-18 and IL-1β were remarkably decreased.5.The expression of NLRP3,caspase-1,GSDMD,IL-18 and IL-1β were markedly increased in H9C2 cell with hypoxia/reoxygenation injury after transfection with AMO-214-3P.Conclusions:The expression of LncRNA KCNQ1OT1 was negatively correlated with the expression of miR-214-3p,and the expression of miR-214-3p was negatively correlated with the degree of pyroptosis during hypoxia/reoxygenation injury in H9C2 cells.Part Ⅳ LncRNAKCNQ1OT1 acts as a ceRNA to mediate pyroptosis in H9C2 cells with hypoxia/reoxygenation injury via sponging miR-214-3p.Objectives:To clarify whether LncRNA KCNQ10T1 acts as a ceRNA to mediate pyroptosis in H9C2 cells with hypoxia/reoxygenation injury via sponging miR-214-3p.Methods:We divided H9C2 cells into 3 groups:small interfering RNA against LncRNA KCNQ1OT1+hypoxia/reoxygenation injury group(si-KCNQ10T1+H/R),small interfering RNA against LncRNA KCNQ1OT1+down-regulated expression of miR-214-3p+hypoxia/reoxygenation injury group(si-KCNQ1OT1+AMO-214-3P+H/R)and down regulated expression of miR-214-3p+hypoxia/reoxygenation injury group(AMO-214-3P+H/R).Cell counting kit-8 was used to detect the cell survival rate.Western blot was used to detect the expression of NLRP3,caspase-1,GSDMD,IL-18 and IL-1β.Results:1.The cell survival rate in si-KCNQ1OT1 AMO-214-3P+H/R group was lower as compared with si-KCNQ1OT1+H/R group,whereas it was higher as compared with AMO-214-3P+H/R group(P<0.05).2.The results of western blot showed that the protein expression of NLRP3,caspase-1,GSDMD,IL-18 and IL-1β in si-KCNQ1OT1+AMO-214-3P+H/R group were significantly increased as compared with si-KCNQ1OT1+H/R group(P<0.05).However,the protein expression of NLRP3,caspase-1,GSDMD,IL-18 and IL-1β in si-KCNQ1OT1+AMO-214-3P+H/R group were significantly decreased as compared with AMO-214-3P+H/R group(P<0.05).Conclusions:LncRNA KCNQ1OT1 might act as a ceRNA to mediate pyroptosis in H9C2 cells with hypoxia/reoxygenation injury via sponging miR-214-3p. |
PDF Full Text Request