Figure 7: Experimental validation of regulatory interaction between miR-29b and NREP.

(a) Association between miR-29b and NREP across cancer types. Bottom, conserved predicted miR-29 target site in NREP, Watson-Crick and wobble (G•U) base pairs are highlighted. (b) Relationship between miR-29b and NREP expression in tumor and representative normal samples in four different cancer types. (c) NREP mRNA expression 24 h after transfection with miR-29b mimic (n = 2 biological replicates, mean ± range) and two different control hairpins (n = 4 biological replicates, mean ± s.e.m.) in cancer cell lines. (d) Relative NREP mRNA expression 24 h after transfection with miR-29b locked nucleic acid (LNA) anti-miR (n = 2, mean ± range) and two control LNAs (n = 4 biological replicates, mean ± s.e.m.). (e) miR-29b expression 24 h after transfection with two different NREP siRNAs (n = 4 biological replicates, mean ± s.e.m.) and two different random control siRNAs (n = 4 biological replicates, mean ± s.e.m.). (f) Putative double-negative feedback loop between the miR-29 family of miRNAs and NREP, which is predicted to impact the active DNA demethylation pathway.