Similarly to human hepatocellular carcinoma, tumors are characterized by a further increase in miR-221 expression and a concomitant inhibition

of its target protein-coding genes (i.e., cyclin-dependent kinase inhibitor [Cdkn]1b/p27, Cdkn1c/p57, and B-cell lymphoma 2–modifying factor). To validate the tumor-promoting effect of miR-221, we showed that in vivo delivery of anti-miR-221 find more oligonucleotides leads to a significant reduction of the number and size of tumor nodules. Conclusions: This study not only establishes that miR-221 can promote liver tumorigenicity, but it also establishes a valuable animal model to perform preclinical investigations for the use of anti-miRNA approaches aimed at liver cancer therapy. (HEPATOLOGY 2012;56:1025–1033) Several studies revealed that the expression of microRNAs (miRNAs) is deregulated in human hepatocellular carcinoma (HCC), in comparison with non-neoplastic liver tissues, as reviewed recently.1

Trametinib Among these, microRNA-221 (miR-221) emerged as consistently up-regulated. In HCC, miR-221 is up-regulated in approximately 70%-80% of cases.2 Its up-regulation in glioblastoma, pancreatic, kidney, bladder, colon, stomach, prostate, and thyroid cancer strengthened its importance in tumorigenesis.2-11 The hypothesized tumor-promoting activity was supported by functional and molecular evidence. Forced expression of miR-221 in HCC cells could induce an increase in growth, proliferation, migration, and invasion capabilities in vitro.2, 10, 12 Conversely, anti-miR-221 oligonucleotides could inhibit in vitro growth DNA Damage inhibitor of liver cancer cells.13 Importantly, the promotion of tumor progression in vivo and the shortening of animal survival was observed when miR-221 was introduced into c-myc-immortalized P53−/− liver progenitor cells, which were implanted into irradiated nude mice.13 Surprisingly, the almost identical miR-222 miRNA, which shares the same seed sequence of miR-221, did not accelerate tumorigenesis in this model system. At the molecular

level, miR-221 was shown to affect several cancer pathways by modulating multiple gene targets, which included the cyclin-dependent kinase inhibitors CDKN1B/p277,11 and CDKN1C/p57,2,10 the pro-apoptotic protein B-cell lymphoma 2-modifying factor (BMF),14 the inhibitor of the phosphoinositide 3-kinase pathway phosphatase and tensin homolog (PTEN),12 the DNA damage-inducible transcript 4 (DDIT4), a tumor suppressor that modulates kinase activity of mammalian target of rapamycin (mTOR),13 the tissue inhibitor of metalloproteinase 3 (TIMP3).12 From a clinical point of view, it was shown that higher levels of miR-221 in HCC correlated with higher tumor stage and metastasis15 and were associated with multifocal tumors and a shorter time to recurrence after surgical treatment.