Research Paper Volume 10, Issue 12 pp 3774—3793

A CREB1/miR-433 reciprocal feedback loop modulates proliferation and metastasis in colorectal cancer

Figure 5. CREB1 transactivated the expression of miR-433. (a) UCSC aligned DBTSS, JASPAR, CONSITE and LASAGNA databases demonstrated that CREB1 could bind to the promotor region of miR-433. (b) The predicted binding sites and mutation sequences are presented. (c-d) Downregulation of miR-433-3p was observed when CREB1 was silenced with siRNA at the mRNA and protein level in LoVo and RKO cells through real-time PCR and western blot. (e-f) The expression of miR-433-3p was subsequently upregulated after overexpression of CREB1 in LoVo and RKO cells by qRT-PCR and western blot. (g) ChIP-qPCR assay indicated that anti-CREB1 enriched much more DNA fragment which contains putative CREB1 binding site on the miR-433 promotor relative to IgG. (h-k) Luciferase assays confirmed the specific targeting relationship between CREB1 and the miR-433 promotor (pmirGlo, empty luciferase reporter vector; wt pmirGlo-mir433, wild-type luciferase reporter plasmid of miR-433 promotor containing putative CREB1 binding site; mut pmirGlo-mir433, luciferase reporter plasmid of miR-433 promotor which mutated putative CREB1 binding site; LV5-NC, lentivirus package of empty vector; LV5-CREB1, lentivirus package of CREB1 overexpressing plasmid). *, p<0.05; **, p<0.01; ***, p<0.001.