3A). Then, a target prediction program, miRanda (http:// www.microrna.org), was used to predict and identify miRNAs that possibly target the endogenous SIRT7 in HCC. From this,

we were able to identify five miRNAs (miR-125a-5p, 125b, 148a, 152, and 193a-3p) that are significantly down-regulated in HCC (Fig. 3B). To confirm the repression of these miRNAs in HCC, quantitative real-time polymerase chain reaction (qRT-PCR) analysis for five miRNAs in Hep3B and SNU-449 cells was performed, and the results compared with that of THLE-3, a normal hepatic liver cell line (Fig. 3C). As expected, the expressions of these five miRNAs were repressed in both Hep3B and SNU-449 cells with some variations. Next, buy C646 to determine whether SIRT7 is selectively regulated by these miRNAs by way of direct interaction with the 3′-untranslated region (UTR) of SIRT7 mRNA, we cloned the 3′-UTR of SIRT7 into click here a reporter vector linking luciferase open reading frame downstream to generate psiCHECK2-SIRT7_3′-UTR

wildtype (psiCHECK2-SIRT7-wt). We also cloned 3′-UTR of random mutation sequences of the SIRT7 gene to generate a mutant-type (psiCHECK2-SIRT7-mt) reporter vector (Supporting Fig. 4A). Then each vector was cotransfected with these miRNAs into Hep3B and SNU-449 cells. The results of dual-luciferase reporter assays of psiCHECK2-SIRT7-wt plasmid with five miRNAs were compared with that of psiCHECK2-SIRT7-mt and are depicted as bar graphs (Fig. 4A,B). It was found that miR-125a-5p, miR-125b, miR-148a, and miR-152 were able to suppress reporter gene activity in both Hep3B and SNU-449 cells, whereas miR-193a-3p had no 上海皓元 effect, therefore indicating that these four miRNAs are able to regulate SIRT7 expression in HCC cells in vitro. Thus, we assessed whether ectopic expression of these five miRNAs mimics the effects of SIRT7 knockdown by siRNA directed against SIRT7 in liver cancer cells. Note that a high level of miRNA expression was detected in both Hep3B and SNU-449 cells after ectopic transfection of five miRNAs (Fig. 4C,D). Consistent

with the results of the luciferase assays, miR-125a-5p, miR-125b, miR-148a, and miR-152 were able to suppress endogenous SIRT7 expression, as SIRT7 siRNA did in both Hep3B and SNU-449 cells (Fig. 4E,F). However, it was found that only miR-125a-5p, miR-125b selectively recovered p21WAF1/Cip1 and suppressed cyclin D1, as SIRT7 siRNA did in both Hep3B and SNU-449 cells. In addition, it was found that expressions of both miR-125a-5p and miR-125b were significantly down-regulated in a large cohort of HCC patients (Supporting Fig. 4B,C). Although it is not clear why miR-148a and miR-152 did not affect these cell cycle proteins, these result suggest that miR-125a-5p and miR-125b are endogenous regulators for SIRT7 in HCC tumorigenesis.