Fig. 5: IKBKBAS competes for binding of miR-4741 with IKKβ mRNA. | Cell Death & Disease

Fig. 5: IKBKBAS competes for binding of miR-4741 with IKKβ mRNA.

From: Novel cytoplasmic lncRNA IKBKBAS promotes lung adenocarcinoma metastasis by upregulating IKKβ and consequential activation of NF-κB signaling pathway

Fig. 5

A The common miRNA that targeted to both IKBKBAS and IKKβ mRNA predicted by RegRNA 2.0, Targetscan Human 7.1, and miRDB. B The relative expression of miR-4741, IKBKBAS, and IKKβ mRNA in A549 and HCC827 cells were assayed by qRT-PCR. C Validation of the effect of miR-4741 inhibitor or mimics on expression of IKKβ in A549 and HCC827 cells by qRT-PCR analysis. D The effect of miR-4741 inhibitor or mimics on expression of IKKβ in A549 and HCC827 cells was assayed by western blot. E Schematic representation of the predicted MREs for miR-4741 on IKBKBAS or IKKβ 3′UTR, and the site mutagenesis design for the luciferase reporter. F Luciferase reporter assay confirmed the interaction between miR-4741 and IKBKBAS or IKKβ 3′UTR in A549 and HCC827 cells. A549 and HCC827 cells were co-transfected with miR-4741 mimic or miR-4741 inhibitor and pMIR-IKBKBAS-wt/mut or pMIR-IKKβ-3′UTR-wt/mut plasmids. miR-NC mimics or miR-NC inhibitor were also transfected as control. Transfected cells were harvested after 48 h and subjected to luciferase activity assay. G The association among IKBKBAS, miR-4741, IKKβ 3′UTR, and AGO2 was ascertained by RNA pull-down assay using A549 cell lysates. Left: Detection of miR-4741 by qRT-PCR in the sample pulled down using biotinylated IKBKBAS and IKBKBAS-AS (negative control) probes; middle: Detection of miR-4741 by qRT-PCR in the sample pulled down using biotinylated IKKβ 3′UTR and IKKβ 3′UTR-AS (negative control) probes; right: Detection of AGO2 by western blot in the sample pulled down using biotinylated IKBKBAS and IKBKBAS-AS (negative control) probes. H The association between IKBKBAS and Ago2 was ascertained by RIP assay using A549 cell lysates with an AGO2 antibody. The anti-SNRNP70 and IgG were used as positive and negative control, respectively. Left: qRT-PCR result; right: RT-PCR result (lane M: marker; lanes 2–4: IKBKBAS fragment, 208 bp; lane 1, 5: U1 snRNA,118 bp). Data show mean ± S.E.M., n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001.

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