MicroRNA-224 Targets SMAD Family Member 4 to Promote Cell Proliferation and Negatively Influence Patient Survival

MicroRNA-224 (miR-224) is frequently over-expressed in liver and colorectal cancers. We and others have previously described the role of miR-224 over-expression in cell proliferation in vitro but we have yet to identify the relevant miR-224 direct target. In this study, we further demonstrated that miR-224 up-regulation promotes cell proliferation using both in vitro assays and in vivo tumor growth models. We systematically screened for high confidence miR-224 targets by overlapping in silico predicted targets from multiple algorithms and significantly down-regulated genes in miR-224-expressing cells from whole genome expression microarrays. A total of 72 high confidence miR-224 targets were identified and found to be enriched in various cancer-related processes. SMAD family member 4 (SMAD4) is experimentally validated as the direct cellular target through which miR-224 promotes cell proliferation. The clinical relevance of our experimental observations was supported by a statistically significant inverse correlation between miR-224 and SMAD4 transcript expression in tumor versus paired adjacent non-tumorous tissues from HCC patients (p<0.001, r= -0.45, R(2) =0.122). Furthermore, miR-224 up-regulation and SMAD4 down-regulation is significantly associated with poorer patient survival (p<0.05). In summary, miR-224/SMAD4 pathway is a clinically relevant pathway to provide new insights in understanding HCC. (191 words).