MicroRNA-23a downregulates the expression of interferon regulatory factor-1 in hepatocellular carcinoma cells.
Interferon regulatory factor-1 (IRF-1) is a tumor-suppressor gene induced by interferon-γ (IFNγ) and plays an important role in the cell death of hepatocellular carcinoma (HCC). HCC tumors evade death in part by downregulating IRF-1 expression, yet the molecular mechanisms accounting for IRF-1 suppression in HCC have not yet been characterized. Previous studies have shown that microRNA-23a (miR-23a) can suppress apoptosis by targeting IRF-1. Therefore, we hypothesized that miR-23a promotes HCC growth by downregulating IRF-1. For the in vivo studies, 7 cases of resected HCC and adjacent liver samples were analyzed. For the in vitro studies, IRF-1 mRNA and protein were examined in HepG2 and Huh-7 HCC cells after IFNγ stimulation by real-time PCR and western blotting, respectively. To determine the role of miR-23a in regulating IRF-1, HepG2 cells were transfected with an miR-23a mimic or inhibitor, and IRF-1 expression was examined. Binding of miR-23a was assessed by cloning the 528-bp human IRF-1 3'-untranslated region (3'UTR) into luciferase reporter plasmid pMIR-IRF-1-3'UTR. The results showed that IRF-1 mRNA expression was downregulated in the human HCC tumor tissues compared to that in the adjacent background liver tissues. IFNγ-induced IRF-1 protein was less in the HepG2 tumor cells compared to that in the primary human hepatocytes. miR-23a expression was inversely correlated with IRF-1, and addition of the miR-23a inhibitor increased basal IRF-1 mRNA and protein. Likewise, the miR-23a mimic downregulated IFNγ-induced IRF-1 protein expression, while the miR-23a inhibitor increased IRF-1. Furthermore, the miR-23a mimic repressed IRF-1-3'UTR reporter activity, while the miR-23a inhibitor increased the reporter activity. These results demonstrated that IRF-1 expression is downregulated in human HCC tumors compared to that noted in the background liver. miR-23a downregulates the expression of IRF-1 in HCC cells, and the IRF-1 3'UTR has an miR‑23a binding site that binds miR-23a and decreases reporter activity. These findings suggest that the targeting of IRF-1 by miR-23a may be the molecular basis for IRF-1 downregulation in HCC and provide new insight into the regulation of HCC by miRNAs.
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Yihe
Y
Yan
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Zhihai
Z
Liang
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Qiang
Q
Du
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Muqing
M
Yang
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.
David A
DA
Geller
D020413:3' Untranslated Regions / Q000235:genetics; D017209:Apoptosis / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D045325:HCT116 Cells; D056945:Hep G2 Cells; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000235:genetics*; D007371:Interferon-gamma / Q000235:genetics; D008113:Liver Neoplasms / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection / Q000379:methods
D020413:3' Untranslated Regions; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; C552430:MIRN23a microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D007371:Interferon-gamma