2D). Consistent with western blotting experiments, qRT-PCR experiments showed that TARDBP regulates expression of only PFKP in SK-Hep1 cells (Fig. 2E). Because TARBDP regulated expression of many glycolysis genes, including PFKP, in multiple HCC cells, we determined the effect of depletion of TARDBP in metabolic response. Glucose uptake of SK-Hep1 cells was significantly reduced by silencing of TARDBP expression (Fig. 3A,B). Furthermore, silencing of TARDBP expression resulted in a decrease in lactate production and ATP levels, indicating a decrease of glycolysis (Fig. 3B). Thus, our findings strongly support the proposed

roles of TARDBP in HCC cell growth through regulation of glucose and energy metabolism. We next attempted HDAC inhibition to determine the molecular mechanism of how TARDBP regulates PFKP expression. Given that the best-known function of TARDBP is RNA processing as an RNA-binding protein,21 we examined whether TARDBP directly interacts with the messenger RNA (mRNA) of PFKP. However, analysis of RNA immunoprecipitation (IP) data

with anti-TARDBP antibody (Ab)21 failed to demonstrate interaction of TARDBP with PFKP mRNA (Supporting Fig. 2), suggesting that TARDBP likely regulates PFKP by other mechanisms. Because TARDBP positively regulates expression of PFKP and also functions as a transcription repressor,22 http://www.selleckchem.com/JNK.html we hypothesized that PFKP could be negatively regulated by intermediate regulators that are, in turn, directly suppressed by TARDBP. Recent studies showed that TARDBP is involved in regulation of miRNAs,23, 24 suggesting that miRNAs might be good candidates for intermediaries between TARDBP and PFKP. To identify such intermediary regulators, we explored target miRNAs that can suppress PFKP based on sequence alignment medchemexpress (Fig. 4A). Sequence analysis with the starBase database25 revealed that 26 miRNAs contain direct binding sequences for the PFKP 3′ untranslated region (UTR) (Supporting Table 1). Interestingly, three

all-independent prediction programs (target Scan, picTarm and miRanda) predicted the miR-520 and miR-302 family as major regulatory miRNAs for PFKP.26 Because previous studies showed that miR-520b and miR-520e can inhibit cancer cell growth,27-29 we next tested whether inhibition of cell growth by miR-520 is mediated by regulation of PFKP expression. When SK-Hep1 cells were treated with miR-520a-3p, miR-520b, and miR-520e (hereafter miR-520a/b/e), expression of PFKP was significantly down-regulated (Fig. 4B), suggesting that PFKP might be a direct target of miR-520a/b/e. However, expression of other glycolysis genes were not significantly altered by miR-520a/b/e (Supporting Fig. 3), suggesting that these miRNAs regulate glycolysis mainly through inhibition of PFKP.