RESUMO
Hepatocyte nuclear factor 4α (HNF4α) is a critical factor for hepatocyte differentiation. HNF4α expression is decreased in hepatocellular carcinoma (HCC), which suggests a role in repression of hepatocyte dedifferentiation. In the present study, hepatic expression of HNF4γ was increased in liver-specific Hnf4a-null mice. The HNF4γ whose expression was increased contained two variants, a known short variant, designated HNF4γ1, and a novel long variant, designated HNF4γ2. HNF4G2 mRNA was highly expressed in small intestine, and the transactivation potential of HNF4γ2 was the strongest among these variants, but the potential of HNF4γ1 was the lowest. Cotransfection experiments revealed that HNF4γ1 repressed HNF4α- and HNF4γ2-dependent transactivation, while HNF4γ2 promoted HNF4α-dependent transactivation. HNF4γ1 and HNF4γ2 were able to bind to the HNF4α binding sites with an affinity similar to that of HNF4α. Furthermore, HNF4γ2, but not HNF4γ1, robustly induced the expression of typical HNF4α target genes to a greater degree than HNF4α. Additionally, HNF4γ2 suppressed proliferation of hepatoma cells as well as HNF4α and HNF4γ1 did, and HNF4γ2 induced critical hepatic functions, such as glucose and urea production, and cytochrome P450 1A2 activity more strongly than HNF4α and HNF4γ1 did. These results indicate that HNF4γ2 has potential for redifferentiation of HCC and thus may be explored as a target for HCC therapy.
Assuntos
Variação Genética/genética , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/metabolismo , Fígado/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CACO-2 , Carcinoma Hepatocelular/genética , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HT29 , Células HeLa , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células PC-3 , RNA Mensageiro/genética , Alinhamento de SequênciaRESUMO
Hepatocyte nuclear factor 4α (HNF4α) controls the expression of liver-specific protein-coding genes. However, some microRNAs are also modulated by HNF4α, and it is not known whether they are direct targets of HNF4α and whether they influence hepatic function. In this study, we found that HNF4α regulates microRNAs, indicated by marked down-regulation of miR-194 and miR-192 (miR-194/192) in liver-specific Hnf4a-null (Hnf4aΔH) mice. Transactivation of the shared miR-194/192 promoter was dependent on HNF4α expression, indicating that miR-194/192 is a target gene of HNF4α. Screening of potential mRNAs targeted by miR-194/192 revealed that expression of genes involved in glucose metabolism (glycogenin 1 (Gyg1)), cell adhesion and migration (activated leukocyte cell adhesion molecule (Alcam)), tumorigenesis and tumor progression (Rap2b and epiregulin (Ereg)), protein SUMOylation (Sumo2), epigenetic regulation (Setd5 and Cullin 4B (Cln4b)), and the epithelial-mesenchymal transition (moesin (Msn)) was up-regulated in Hnf4aΔH mice. Moreover, we also found that miR-194/192 binds the 3'-UTR of these mRNAs. siRNA knockdown of HNF4α suppressed miR-194/192 expression in human hepatocellular carcinoma (HCC) cells and resulted in up-regulation of their mRNA targets. Inhibition and overexpression experiments with miR-194/192 revealed that Gyg1, Setd5, Sumo2, Cln4b, and Rap2b are miR-194 targets, whereas Ereg, Alcam, and Msn are miR-192 targets. These findings reveal a novel HNF4α network controlled by miR-194/192 that may play a critical role in maintaining the hepatocyte-differentiated state by inhibiting expression of genes involved in dedifferentiation and tumorigenesis. These insights may contribute to the development of diagnostic markers for early HCC detection, and targeting of the miR-194/192 pathway could be useful for managing HCC.
Assuntos
Regulação da Expressão Gênica/fisiologia , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , MicroRNAs/metabolismo , Transdução de Sinais/fisiologia , Regiões 3' não Traduzidas/fisiologia , Molécula de Adesão de Leucócito Ativado/biossíntese , Molécula de Adesão de Leucócito Ativado/genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Epirregulina/biossíntese , Epirregulina/genética , Glucosiltransferases/biossíntese , Glucosiltransferases/genética , Glicoproteínas/biossíntese , Glicoproteínas/genética , Fator 4 Nuclear de Hepatócito/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Mutantes , MicroRNAs/genética , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/biossíntese , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
To obtain a high antimalarial activity with neocryptolepine derivatives, modifying and changing the side chains at the C11 position with varying the substituents of an electron-withdrawing or electron-donating nature at the C2 position for a SAR study were executed. Installation of alkylamino and ω-aminoalkylamino groups at the C11 position of the neocryptolepine core was successful. For further variation, the aminoalkylamino substituents were transformed into the corresponding acyclic or cyclic carbamides or thiocarbamides. These side chain modified neocryptolepine derivatives were tested for antimalarial activity against CQR (K1) and CQS (NF54) of Plasmodium falciparum in vitro and for cytotoxicity toward mammalian L6 cells. Among the tested compounds, the compound 17f showed an IC50 of 2.2 nM for CQS (NF54) and a selectivity index of 1400, and 17i showed an IC50 of 2.2 nM for CQR (K1), a selectivity index of 1243, and a resistance index of 0.5.
