As shown in Supporting Fig. 1A, USP28 was detected in HepG2, BEL-7402, and FHCC98 liver tumor cell lines, whereas it was undetectable in normal cell lines. When HepG2 and BEL-7402 cells were transfected with a USP28 siRNA, we noted a subsequent decrease LBH589 mouse of Myc at the protein level but not at the mRNA level (Fig. 5B,C) whereas inhibition of USP28 with MG132 resulted in no changes in Myc protein or mRNA levels (Fig. 5E). We also observed that inhibition of USP28 suppressed the malignant phenotype of HepG2 and BEL-7402 cells in a manner that correlated with their reduced Myc levels (Fig. 5D). Co-IP experiments also confirmed that USP28 directly interacts with Myc in HepG2 cells (Fig. 5F).

These data indicate that miR-363-3p indirectly destabilizes Myc by directly targeting USP28. Among cell lines used in this study, miR-148a-5p

and miR-363-3p express high levels in the normal human hepatocyte cell line HL7702 and human liver tumor cell line PD0325901 supplier FHCC98. To elucidate whether inhibition of miR-148a-5p or miR-363-3p affected cellular properties associated with the malignant phenotype, we inhibited each miRNA using miRNA-specific inhibitors in HL7702 and FHCC98 cells. As shown in Fig. 6A,B, this significantly decreased their miRNA expression and led to increases in their target transcripts and proteins in both cases. It also enhanced several malignant phenotypes (Fig. 6C,D). For example, it promoted G0/G1 to S phase progression in HL7702 and FHCC98 cells (Fig. 6E) and also promoted cell migration (Supporting Fig. 10). Moreover, a significant increase in tumor growth rates were observed when compared with the tumors expressing control miRNA inhibitor in FHCC98 cells (Fig. 6F). These studies show that both gain- and loss-of-function of miR-148a-5p or miR-363-3p affected multiple aspects of the malignant phenotypes in HCCs. In order to investigate whether the Myc-miRNA feedback loop is dysregulated in human HCCs, expression levels of Myc, USP28,

mir-148a-5p, and mir-363-3p were quantified in total RNA derived from two normal human cell lines, four human liver tumor cell lines, 27 hepatocarcinomas, and paired normal hepatic tissues. These Acyl CoA dehydrogenase studies showed increased levels of Myc in 17 of the HCCs, (>1.5 fold change) and increased levels of USP28 in 21 cases. In contrast, mir-148a-5p and mir-363-3p transcripts were reduced in 23 and 11 cases, respectively (Fig. 7A-C). Hence, the Myc-miRNA feedback loop is dysregulated in HCCs. Furthermore, our results generally showed a positive correlation between Myc and USP28 levels, a negative correlation between Myc and mir-148a-5p or mir-363-3p levels, and a negative correlation between USP28 and mir-363-3p levels (Fig. 7A,D). In addition, western blot assays showing Myc or USP28 protein levels were up-regulated in HCC relative to adjacent normal tissues, in human liver tumor cell lines relative to normal human cell lines (Fig. 7E and Supporting Fig. 1A).