Supporting experiments indicated that ectopic expression of miR-148a-5p or miR-363-3p induced a consistent G0/G1 arrest in HepG2 and BEL-7402 cells, but not HL7702 cells (Fig. 2D). Ectopic expression of miR-148a-5p and miR-363-3p also inhibited the migration in HepG2 and BEL-7402 cells (Supporting

Fig. 7). To determine whether mir-148a-5p or mir-363-3p could inhibit tumor growth, HepG2 cells ectopically expressing mir-148a-5p or mir-363-3p were injected into the flanks of nude mice. This resulted in a significant decrease of tumor growth compared with the tumors expressing empty vector (Fig. 2E). Taken together, these data suggest that miRNAs induce cell cycle arrest and inhibit tumor growth in HCC cells. To elucidate JNK pathway inhibitor the molecular mechanism by which both miRNAs induce CX-5461 purchase cell cycle arrest and inhibit tumorigenicity, we performed miRDB, miRanda, miRwalk, and RNAhybrid analyses to identify functional targets of miR-148a-5p and miR-363-3p. These analyses revealed the 3′-UTR of Myc to contain one highly conserved miR-148a-5p binding site from human to Canis familiaris

(Fig. 3A), whereas the 3′-UTR of USP28 mRNA, the ubiquitin protease of Myc, contains one highly conserved from human to Equus caballus and the other nonconserved miR-363-3p binding sites (Fig. 4A). We tested whether Myc or USP28 are direct targets of miR-148a-5p Linifanib (ABT-869) or miR-363-3p, respectively. For this, a GFP reporter assay was employed to detect the potential interaction of each miRNA with the 3′-UTR of targets. The results showed that miR-148a-5p inhibited the GFP expression of a vector containing the predicted miR-148a-5p binding site but not the GFP vector only (Fig. 3B). We also found that miR-363-3p repressed the GFP expression of a vector containing either one or both of the predicted miR-363-3p binding sites, but not the nonconserved

binding site (Fig. 4B). These data supported direct inhibition of Myc by miR-148a-5p and USP28 by miR-363-3p. As expected, ectopic expression miR-148a-5p in HepG2 and BEL-7402 HCC cells resulted in a marked decrease of Myc mRNA and protein and an increase in mir-363-3p. This was further associated with decrease in USP28 mRNA and protein (Fig. 3C,D); ectopic expression miR-363-3p in HepG2 and BEL-7402 cells resulted in a marked decrease of USP28 at both mRNA and protein levels, while in a marked decrease of Myc at protein level but not mRNA level (Fig. 4C,D). Although the 3′-UTR of Myc does not contain predicted binding sites for miR-363-3p, ectopic expression miR-363-3p also led to a marked decrease of Myc protein but not mRNA (Fig. 4C,D). The reduction in Myc protein could be prevented, however, if the cells were exposed to MG132, a proteasome inhibitor (Fig. 5A).