Figure 7. miR-376b targets NFKBIZ in LPS induced septic AKI.

(A) Conserved miR-376b target sequence in the 3′-UTR of NFKBIZ mRNA. (B) Immunofluorescence showing the repressive effect of miR-376b on NFKBIZ expression. BUMPT cells were transfected with 200 nM miR-376b mimic or NC for 24 hours and then treated with or without 10 μg/mL LPS for 8 hours. (C) Immunoblot analysis showing the repressive effect of miR-376b on NFKBIZ expression. BUMPT cells were treated as in B to collect lysate for immunoblot analysis. GAPDH was used as internal control in immunoblot analysis. (D) Immunofluorescence showing the repressive effect of miR-376b on NFKBIZ expression. miR-376b mimic (7 mg/kg) or NC oligonucleotides were delivered to male C57BL/6 mice through tail vein injection 1 day before LPS injection. The mice were then injected intraperitoneally with 10 mg/kg LPS, while control mice were injected with normal saline. Kidney tissues were collected at 24 hours after LPS injection. Scale bar: 100 μm. (E) Immunoblot analysis showing the repressive effect of miR-376b on NFKBIZ expression. Mice were subjected to the same treatment as in D to collect kidney tissues for immunoblot analysis. GAPDH was used as internal control. (F) MicroRNA target reporter assay of Nfkbiz 3′-UTR. The putative miR-376b target sequence of the Nfkbizc 3′-UTR was cloned into the pMIR-REPORT vector. This and empty vector were transfected with miR-376b mimic or NC oligonucleotides to analyze luciferase activity. miR-376b mimic specifically reduced luciferase expression by pMIR-REPORT Nfkbiz 3′-UTR. Quantitative data are expressed as mean ± SD (n = 4 or n = 5, 2-tailed Student’s t test), *P < 0.05.