Figure 5.

Phosphorylated Rb protein promotes the expression of miR-21 and miR-181b in Gr1⁺CD11b⁺ cells during sepsis. Gr1⁺CD11b⁺ cells were isolated from the bone marrow of late septic NFI-A conditional knockout mice and transfected with an NFI-A expression plasmid or an empty vector for 36 h. (a) Ectopic expression of NFI-A restores RB phosphorylation and the miRNA expression. Levels of CDK4, Rb and p-Rb (Ser⁷⁸⁰) proteins were determined by Western blot. The results are representative of three experiments. (b) miRNA-enriched RNA was isolated, and levels of miR-21 and miR-181b expression were measured by RT-qPCR using miR-21 and miR-181b specific assay primer sets. Values were normalized to U6 RNA as an internal control. Data are expressed as mean ± SD (*P < 0.05) of five mice per group and represent one of two experiments. *Compared with vector. (c) Ectopic expression of NFI-A in the absence of RB does not restore the miRNA expression. Gr1⁺CD11b⁺ cells, isolated from the bone marrows of late septic conditional NFI-A knockout mice, were transfected with Rb-specific or scramble (control) siRNAs. After 12 h, cells were washed and transfected with an NFI-A expression plasmid or an empty vector for 24 h. Cell extracts were prepared from portion of the cells and used to determine Rb protein levels by Western blot. The results are representative of two Western blots. The remainder of the cells was used to isolated miRNA-enriched RNA, and levels of miR-21 and miR-181b were analyzed by real-time PCR as in (b). Data are expressed as mean ± SD (*P < 0.05) of five mice per group and represent one of three experiments. cKO: conditional knockout.