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. 2021 Dec 29;82(4):556–570. doi: 10.1158/0008-5472.CAN-21-1446

Figure 5.

Figure 5. Uc003xsl.1 serves as a molecular decoy between NKRF and IL8. A, Schematic images of IL8 promoter. The red frame displays the potential region of NKRF enrichment in the IL8 promoter by ChIP-seq analysis, and the specific primers for following ChIP-PCR analysis are underlined. B and C, ChIP-PCR analyzed NKRF enrichment (B) and P65 enrichment (C) in the IL8 promoter in MDA-MB-231-P3 cells after transfection of control siRNA, Uc003xsl.1 siRNA, P65 siRNA, and NKRF siRNA. D, Fragments and conserved domains of NKRF protein. E and F, RIP assays identified that the NKRF3-D3 fragment specifically binds to Uc003xsl.1 in MDA-MB-231 cells. G, ChIP-PCR analysis revealed that the NKRF3-D3 fragment binds to the IL8 promoter but not the NKRF-DR3H in MDA-MB-231 cells. H, The sequence of the IL8 promoter. Identified binding sites for transcription factors are illustrated (23). Shown is the sequence of oligonucleotides of IL8 promoter WT, P65 mutant, and NRE mutant. Point mutations are underlined. The P65-binding site and NRE are shaded. I, IL8 promoter luciferase activity was detected in MDA-MB-231 cells after transfection of luciferase reporter plasmids containing the IL8 promoter WT, or versions carrying mutations in the NRE (NREmut) or P65-binding site (P65mut). J, IL8 promoter luciferase activity was assessed in MDA-MB-231 cells after cotransfection of luciferase reporter vector containing the WT IL8 promoter and NKRF expression vectors, with or without R3H domain. K, The schematic working model of Uc003xsl.1 overexpression drives the NKRF/NFκB/IL8 axis in TNBC. Uc003xsl.1 upregulated in TNBC could bind to R3H domain of NKRF and directly masks the binding motif of R3H with NRE in NFκB-responsive gene (IL8) promoter, thereby abolishing NKRF repression to IL8 transcriptional activity and finally promoting NFκB/IL8-mediated TNBC metastasis. Error bars, SDs of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Uc003xsl.1 serves as a molecular decoy between NKRF and IL8. A, Schematic images of IL8 promoter. The red frame displays the potential region of NKRF enrichment in the IL8 promoter by ChIP-seq analysis, and the specific primers for following ChIP-PCR analysis are underlined. B and C, ChIP-PCR analyzed NKRF enrichment (B) and P65 enrichment (C) in the IL8 promoter in MDA-MB-231-P3 cells after transfection of control siRNA, Uc003xsl.1 siRNA, P65 siRNA, and NKRF siRNA. D, Fragments and conserved domains of NKRF protein. E and F, RIP assays identified that the NKRF3-D3 fragment specifically binds to Uc003xsl.1 in MDA-MB-231 cells. G, ChIP-PCR analysis revealed that the NKRF3-D3 fragment binds to the IL8 promoter but not the NKRF-DR3H in MDA-MB-231 cells. H, The sequence of the IL8 promoter. Identified binding sites for transcription factors are illustrated (23). Shown is the sequence of oligonucleotides of IL8 promoter WT, P65 mutant, and NRE mutant. Point mutations are underlined. The P65-binding site and NRE are shaded. I, IL8 promoter luciferase activity was detected in MDA-MB-231 cells after transfection of luciferase reporter plasmids containing the IL8 promoter WT, or versions carrying mutations in the NRE (NREmut) or P65-binding site (P65mut). J, IL8 promoter luciferase activity was assessed in MDA-MB-231 cells after cotransfection of luciferase reporter vector containing the WT IL8 promoter and NKRF expression vectors, with or without R3H domain. K, The schematic working model of Uc003xsl.1 overexpression drives the NKRF/NFκB/IL8 axis in TNBC. Uc003xsl.1 upregulated in TNBC could bind to R3H domain of NKRF and directly masks the binding motif of R3H with NRE in NFκB-responsive gene (IL8) promoter, thereby abolishing NKRF repression to IL8 transcriptional activity and finally promoting NFκB/IL8-mediated TNBC metastasis. Error bars, SDs of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.