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. 2018 Feb 14;9(16):13048–13059. doi: 10.18632/oncotarget.24495

Figure 2. miR-20a directly modulates CXCL8 protein levels.

Figure 2

(A–C) miR-20a in situ hybridization on paraplast section of intestinal tissue from healthy individuals (A) and patients with colitis (B) and sporadic cancer (C). miR-20a (blue precipitate) is expressed in the epithelia (indicated by arrows), but also in the stroma between the intestinal crypts (indicated by arrowheads). The latter expression is markedly reduced in colitis and colon cancer. Scale bar 0.5 mm. (D) Sequence alignment of the miR-20a/b and miR-19a/b-1 binding sites in the CXCL8 3′UTR. (E) Luciferase assay for the individual members of the miR17~92 miRNA cluster regulating CXCL8. (F, G) Levels of secreted endogenous CXCL8 are modulated in a concentration dependent manner in miR-20a loss- and gain-of-function studies using antagomirs (F) and mimics (G). Asterisks indicate significance (2-way ANOVA with *p < 0.05) (H) Luciferase assays using the wildtype pMirGLO-CXCL8 3′UTR luciferase construct and one with a mutated miR-20a binding site in the presence and absence of a non-targeting miRNA mimic or miR-20a-5p. Asterisks indicate significance (t-test with *p < 0.05 and **p < 0.01). (I) RNA immuno-precipitation using the miR-Trap system (Takara) in the presence of a miR-20a duplex and as a negative control a miR-1 duplex, which does not have a detectable binding site in the CXCL8 3′UTR. Enrichment scores were calculated. Significance was assessed using t-test (**p < 0.005).