Expression of Annexin A3 in Primary Cultured Parenchymal Rat Hepatocytes and Inhibition of DNA Synthesis by Suppression of Annexin A3 Expression Using RNA Interference

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  • Niimi Shingo
    Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Japan
  • Harashima Mizuho
    Department of Nutrition and Physiology, Nihon University College of Bioresource Sciences
  • Gamou Masaru
    Department of Nutrition and Physiology, Nihon University College of Bioresource Sciences
  • Hyuga Masashi
    Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Japan
  • Seki Taiichiro
    Department of Nutrition and Physiology, Nihon University College of Bioresource Sciences
  • Ariga Toyohiko
    Department of Nutrition and Physiology, Nihon University College of Bioresource Sciences
  • Kawanishi Toru
    Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Japan
  • Hayakawa Takao
    Deputy Director General, National Institute of Health Sciences, Japan

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Annexin A3 is a member of the lipocortin/annexin family, which binds to phospholipids and membranes in a Ca2+-dependent manner. Although annexin A3 has various functions in vitro, its cellular significance is completely unknown. Annexin A3 is not found in rat liver in vivo. In the present study, we investigated the expression of annexin A3 in primary cultured parenchymal rat hepatocytes. Annexin A3 protein was detected in 48-h, but not 2.5-h, cultured hepatocytes using Western blot analysis. The annexin A3 level further increased after an additional 24 h of culture. Annexin A3 mRNA was not detected in 2.5-h cultured hepatocytes but was detected 22 h after the start of culture by RT-PCR analysis, reaching a maximum value after 48 h of culture. To define the role of Annexin A3 in DNA synthesis, RNA interference was used to reduce annexin III gene expression in hepatocytes. The transfection of small interfering RNAs targeting annexin A3 in the hepatocytes reduced the corresponding mRNA and protein expression by approximately 80% and more than 90%, respectively, at 24 h after transfection. In the annexin A3 small interfering RNAs-transfected cells, DNA synthesis, as assessed by [3H]thymidine incorporation, decreased by approximately 70% not only in the control cultures, but also in the hepatocyte growth factor- or epidermal growth factor-treated cells. These findings show that annexin A3 is expressed in primary cultured parenchymal rat hepatocytes and that the suppression of annexin A3 expression using RNA interference inhibits DNA synthesis.

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