Non-target Genes Regulate miRNAs-Mediated Migration Steering of Colorectal Carcinoma

MicroRNAs (miRNAs) trigger a two-layer regulatory network directly or through transcription factors and their co-regulators. Unlike miR-375 , the role of miR-145 and miR-224 in inhibiting or driving cancer cell migration is controversial. This study is a step towards addressing the potential of miR-375 , miR-145 and miR-224 expression modulation to inhibit colorectal carcinoma (CRC) cells migration in vitro through regulation of non-target genes VEGFA , TGFβ1 , IGF1 , CD105 and CD44 . Transwell migration assay results revealed a significant subdue of migration ability of cells transfected with miR-375 and miR-145 mimics and miR-224 inhibitor. Real time PCR data showed that expression of VEGFA , TGFβ1 , IGF1 , CD105 and CD44 was downregulated as a consequence of exogenous re-expression of miR-375 and inhibition of miR-224 . On the other hand, ectopic expression of miR-145 did not affect VEGFA , TGFβ1 and CD44 expression, while it elevated CD105 and suppressed IGF1 expression. MAP4K4, a predicted target of miR-145, was validated as a target that could play a role in miR-145 -mediated regulation of migration. At mRNA level, no change was observed in expression of MAP4K4 in cells with restored expression of miR-145 , while western blotting analysis revealed a 25% reduction of protein level. By applying luciferase reporter assay, a significant decrease in luciferase activity was observed, supporting that miR-145 directly target 3’ UTR of MAP4K4 . The study highlighted the involvement of non-target genes VEGFA , TGFβ1 , IGF1 , CD105 and CD44 in mediating anti- and pro-migratory effect of miR-375 and miR-224 , respectively, and validated MAP4K4 as a direct target of anti-migratory miR-145 .