Figure 2.

lncRNA AK085865 Regulates ILF2-ILF3 Complex Functions and miRNA Biogenesis

BMDMs were prepared from AK085865^−/− (KO) and wild-type (WT) mice. (A) ILF2 and ILF3 protein levels were detected by western blot. β-actin was used as a loading control. (B) Coimmunoprecipitation was performed using antibodies against ILF2 for the pull-down assay, and the proteins interacting with ILF2 were eluted and quantified by western blot. (C) Volcano plots of miRNAs differentially expressed in BMDMs from AK085865^−/− and WT mice. Upregulated and downregulated genes are shown as red and blue dots, respectively; n = 3 for each group. (D) Validation of the selected miRNAs differentially expressed in BMDMs from AK085865^−/− and WT mice (n = 6–8/group). (E) Knockdown of ILF2 decreases pri-miRNA and increases mature miRNA. BMDMs from WT mice were transfected with ILF2 siRNAs, and RNAs were isolated and analyzed for the amount of pri-miRNAs and mature miRNAs by qRT-PCR with specific primers. GAPDH and snRNA U6 were used as an internal control and for normalization of the data. (F and G) Knockdown of ILF2 rescues the accumulation of pri-miRNA processing mediated by AK085865 deletion. BMDMs from AK085865^−/− mice were transfected with ILF2 siRNAs. RNAs were isolated and analyzed for (F) the amount of pri-miRNAs and (G) mature miRNAs by qRT-PCR with specific primers. The data are expressed as the mean ± SEM of three independent experiments. NC, negative control. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.