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. 2017 Sep 29;24(12):2199–2209. doi: 10.1038/cdd.2017.151

Figure 8.

Figure 8

miR-148a-3p/5p directly targets several upstream regulators of NF-κB and STAT3. (a) Schematic of human GP130, IKKα, IKKβ, TNFR2 and ILR1 3′-untranslated regions (UTRs). The predicted miR-148a-3p/5p-binding sites, which are conserved between human and mouse, are indicated. (b) miR-148a deletion upregulates mRNA level of miR-148a-3p/5p targets. The above targets mRNA levels, normalized for glyceraldehyde 3-phosphate dehydrogenase, were measured by reverse transcription quantitative-PCR (RT-qPCR) in RNA purified from colon tissues of WT and miR-148a KO mice (n=3). (c) miR-148a deletion enhances the protein levels of miR-148a-3p/5p targets. Immuno-blotting for the above targets proteins in colon tissues from WT and miR-148a KO mice. Each lane represents an individual mouse. (d) Luciferase activity of the reporter vector containing the WT or miR-148a-3p and 5p binding mutant 3′-UTR of miR-148a-3p/5p targets was determined after co-transfection with control, Pri-miR-148a or miR-148a-3p sponge expressing vectors. (e) RT-qPCR analysis was performed to detect miR-148a-3p/5p targets RNA level incorporated into RISC derived from colon tissues of WT and miR-148a KO mice. (f) miR-148a-3p/5p targets expression in human IBD and normal colon tissues. (g) The expression of GP130, IKKα, IKKβ, TNFR2 and ILR1 is negatively correlated with miR-148-3p and 5p, respectively. Each point is an individual IBD or normal colon sample. r, Spearman correlation coefficient. (h) The expression of miR-148a-3p/5p and their targets in 323 CRC samples from TCGA data set is shown in a clustered heatmap. A vertical branch shows the expression pattern of the selected miRNAs or genes in each CRC sample from the TCGA data set. (i) Schematic summary. In normal epithelium, miR-148a directly targets GP130, IL1R1, IKKα, IKKβ and TNFR2 transcripts and thus inhibits NF-κB and STAT3 signaling pathways. miR-148a suppression activates NF-κB and STAT3 to promote colitis and colitis-associated tumorigenesis. In a negative feedback loop, NF-KB inhibits miR-148a expression by recruiting DNMT3A to methylate the miR-148a promoter. Data present mean±S.D. in panels (b, d and e). *P<0.05, **P<0.01, ***P<0.001