ABSTRACT
microRNA-199a (miR-199a) is a highly conserved miRNA, always deregulated in numerous human tumors. The results of microarray analysis indicated that miR-199a was frequently downregulated in hepatocellular carcinoma (HCC). The expression levels of miR-199a in 11 pairs of matched HCC neoplastic and adjacent non-neoplastic tissues, 5 HCC cell lines and liver cell line L02 were examined by quantitative real-time PCR analysis. We found miR-199a was significantly down-regulated in HCC tissues when compared with pair-matched adjacent non-tumor tissues. We also found the expression level of miR-199a was also substantially decreased in several human HCC cell lines including SMMC-7721, BEL-7402, BEL-7701, MHCC97H, and HepG2. To investigate the role of miR-199a in tumorigenesis, we developed a lentiviral vector for the expression of pre-miR-199a (Lenti-miR-199a). Lenti-miR-199a inhibited HCC cell proliferation in vitro and in vivo. Compared to parental cells or cells transfected with a control vector, the overexpression of microRNA-199a in the HCC cell lines HepG2 stably was showed to reduce cell proliferation in vitro and in vivo. Luciferase reporter assay revealed the regulation of miR-199a on 3'-UTR of HIF-1α. Further investigation confirmed that miR-199a significantly reduced the endogenous protein level of HIF-1α in hypoxia. MiR-199a inhibits cell proliferation in vitro and in vivo partly through down-regulation of HIF-1α in human HCC. Thus, these studies provide an important new insight into the pathogenesis of human HCC and it may open a new perspective for the development of effective gene therapy for human HCC.
Subject(s)
Carcinoma, Hepatocellular/pathology , Lentivirus/genetics , Liver Neoplasms/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , 3' Untranslated Regions , Animals , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation , Genetic Vectors/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Mice, Nude , Transplantation, HeterologousABSTRACT
The 70kDa ribosomal S6 kinase 1 (p70S6K1), a downstream target of phosphoinositide 3-kinase (PI3K) and ERK mitogen-activated protein kinase (MAPK), is an important regulator of cell cycle progression, and cell proliferation. Recent studies indicated an important role of p70S6K1 in PTEN-negative and AKT-overexpressing tumors. However, the mechanism of p70S6K1 in tumor angiogenesis remains to be elucidated. In this study, we specifically inhibited p70S6K1 activity in ovarian cancer cells using vector-based small interfering RNA (siRNA) against p70S6K1. We found that knockdown of p70S6K1 significantly decreased VEGF protein expression and VEGF transcriptional activation through the HIF-1alpha binding site at its enhancer region. The expression of p70S6K1 siRNA specifically inhibited HIF-1alpha, but not HIF-1beta protein expression. We also found that p70S6K1 down-regulation inhibited ovarian tumor growth and angiogenesis, and decreased cell proliferation and levels of VEGF and HIF-1alpha expression in tumor tissues. Our results suggest that p70S6K1 is required for tumor growth and angiogenesis through HIF-1alpha and VEGF expression, providing a molecular mechanism of human ovarian cancer mediated by p70S6K1 signaling.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Neovascularization, Pathologic/metabolism , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/pathology , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Vascular Endothelial Growth Factor A/biosynthesis , Cell Line, Tumor , Female , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Neovascularization, Pathologic/genetics , Ovarian Neoplasms/genetics , RNA, Small Interfering/genetics , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Transcriptional Activation , Vascular Endothelial Growth Factor A/antagonists & inhibitorsABSTRACT
OBJECTIVE: To investigate the effect of 2-phenoxyethanol on potency of Sabin inactivated poliomyelitis vaccine (IPV). METHODS: Sabin IPV samples containing 5 mg or 7 mg 2-phenoxyethanol each dosage respectively were placed separately at 4 degrees C, 37 degrees C for 2 days and 7 days. D-antigen contents were tested with ELISA method. Then neutralizing antibodies in mice and guinea pigs were detected. The safety experiment was performed according to unusual toxicity test of China requirement for biological product. RESULTS: After addition of 2-phenoxyethanol, the I, II, and III D-antigen contents of Sabin IPV did not change. The antibody levels in mice and guinea pigs were not different between experimental group and control group. Animals were safe during observation period. CONCLUSION: 2-Phenoxyethanol had no effect on potency and safety of Sabin IPV. It can be used as antiseptic for Sabin IPV.
