The Mechanism Of MiR-155 Regulating LPS-induced Podocyte Pyroptosis By Targeting The Expression Of NLRP1 Inflammasome

Part I The expression of miR-155 in LPS-induced podocyte pyroptosisObjective:To evaluate the modeling effect and detect the expression level of miR-155 in the podocyte pyroptosis model by establishing the model of LPS-induced podocyte pyroptosis.Methods:(1)Mouse podocyte MPC5 were was cultured in vitro and mycoplasma was detected.(2)The m RNA and protein expression levels of Caspase-1,IL-1β and IL-18 were detected after treated with different concentrations of LPS(0 μg/ml,1 μg/ml,5 μg/ml,10μg/ml,20 μg/ml)after 24 h intervention of podocytes.(3)The m RNA and protein expression levels of Caspase-1,IL-1β and IL-18 were detected after treated with 10 μg/ml LPS for different time(0 h,12 h,24 h,36 h and 48 h).(4)The expression of miR-155 in LPS-induced podocyte pyroptosis was detected.(5)miR-155 Over-Expression and miR-155 Knock-Down were transfected with lentivirus and stable strains were screened.Results:(1)The mouse podocyte MPC5 used in this experiment is free of mycoplasma contamination.(2)The m RNA and protein expressions of Caspase-1,IL-1β and IL-18 were upregulated after treated with different concentrations of LPS(0 μg/ml,1 μg/ml,5 μg/ml,10μg/ml,20 μg/ml)for 24 h after intervention.LPS with concentrations of 5 μg/ml,10 μg/ml,and 20 μg/ml can significantly increase the relative expression of miR-155.(3)The expression levels of Caspase-1,IL-1β,IL-18 m RNA and protein were up-regulated in different degrees after 10 μg/ml LPS was applied to podocytes for different times(0 h,12 h,24 h,36 h,48 h),and the relative expression of miR-155 was higher than that of the normal control group at 12 h and 24 h.(4)The stable strains of miR-155 Over-Expression and miR-155 Knock-Down were successfully screened.Conclusion:LPS can induce podocyte pyroptosis,and the expression level of miR-155 is up-regulated in podocyte pyroptosis model.Part II The mechanism of miR-155 regulating LPS-induced podocyte pyroptosis by targeting the expression of NLRP1 inflammasomeObjective:To explore whether miR-155 regulates LPS-induced podocyte pyroptosis by targeting the expression of NLRP1 inflammasome.Methods:(1)miR-155 target genes were screened by bioinformatics website and verified by luciferase reporter gene assay and Western Blot;(2)LPS induces the pyroptosis of podocytes,miR-155 Over-Expression stable strains and miR-155 Knock-Down stable strains,and the expressions of NLRP1,Caspase-1,IL-1β,IL-18 and IL-18 BP were detected at the transcription level and protein level.(3)Immunofluorescence was used to observe the expression of NLRP1,Caspase-1 and IL-18 in podocytes after LPS treatment.Results:(1)There were binding sites between miR-155 and the 3’UTR of NLRP1 gene,luciferase reporter gene assay confirmed that miR-155 could bind to NLRP1,and Western Blotting showed that the over-expression of miR-155 could inhibit the expression of NLRP1 protein.(2)Compared with the LPS group,the over-expression of miR-155 can inhibit the expression level of NLRP1,Caspase-1,IL-1β,and IL-18,while knockdown the the expression of miR-155 can up-regulate the expression level of NLRP1,Caspase-1,IL-1β and IL-18.(3)Immunofluorescence results showed that the fluorescence intensity of NLRP1,Caspase-1 and IL-18 was significantly enhanced in podocyte pyroptosis in the LPS+miR-155Knock-Down group.Conclusion:miR-155 can inhibit the expression of NLRP1 protein by binding the 3’UTR of NLRP1 gene,reduce the expression of Caspase-1,IL-1β and IL-18,MiR-155 may be involved in the regulation of LPS-induced podocyte pyroptosis by targeting NLRP1 inflammasome.