MicroRNA-433 Inhibits Liver Cancer Cell Migration by Repressing the Protein Expression and Function of cAMP Response Element-binding Protein

Background: Although its expression is altered in several human diseases, the function of miR-433 is unknown. Results: miR-433 inhibits HCC cell migration by repressing CREB1 3′-UTR activity and protein expression. Conclusion: CREB1 is the first identified target of miR-433 in liver cancer. Significance: This finding has broad implications given the role of CREB1 in various cancers, hepatic glucose metabolism, and liver circadian clock regulation. We show for the first time that potent microRNA-433 (miR-433) inhibition of expression of the cAMP response element-binding protein CREB1 represses hepatocellular carcinoma (HCC) cell migration. We identified a miR-433 seed match region in human and mouse CREB1 3′-UTRs. Overexpression of miR-433 markedly decreased human CREB1 3′-UTR reporter activity, and the inhibitory effect of miR-433 was alleviated upon mutation of its binding site. Ectopic expression of miR-433 reduced CREB1 protein levels in a variety of human and mouse cancer cells, including HeLa, Hepa1, Huh7, and HepG2. Human CREB1 protein levels in highly invasive MHCC97H cells were diminished by expression of miR-433 but were induced by miR-433 antagomir (anti-miR-433). The expression of mouse CREB1 protein negatively correlated with miR-433 levels in nuclear receptor Shp −/− liver tissues and liver tumors compared with wild-type mice. miR-433 exhibited a significant repression of MHCC97H cell migration, which was reversed by anti-miR-433. Overexpressing miR-433 inhibited focus formation dramatically, demonstrating that miR-433 may exert a tumor suppressor function. Knockdown of CREB1 by siRNAs impeded MHCC97H cell migration and invasion and antagonized the effect of anti-miR-433. Interestingly, CREB1 siRNA decreased MHCC97H cell proliferation, which was not influenced by anti-miR-433. Overexpressing CREB1 decreased the inhibitory activity of miR-433. The CpG islands surrounding miR-433 were hypermethylated, and the DNA methylation agent 5′-aza-2′-deoxycytidine, but not the histone deacetylase inhibitor trichostatin A, drastically stimulated the expression of miR-433 and miR-127 in HCC cells. The latter is clustered with miR-433. The results reveal a critical role of miR-433 in mediating HCC cell migration via CREB1.