ABSTRACT
Primary human hepatocytes (PHHs) are the 'gold standard' for investigating hepatitis B virus (HBV) infection and antiviral drugs. However, poor availability, variation between batches and ethical issues regarding PHHs limit their applications. The discovery of human sodium taurocholate cotransporting polypeptide (hNTCP) as a functional HBV receptor has enabled the development of a surrogate model to supplement the use of PHHs. In the present study, the evolutionary distance of seven species was assessed based on singlecopy homologous genes. Based on the evolutionary distance and availability, PHHs and primary rabbit hepatocytes (PRHs) were isolated and infected with hNTCPrecombinant lentivirus, and susceptibility to HBV infection in the two cell types was tested and compared. In addition, HBV infection efficiency of hNTCPexpressing PPHs with pooled HBVpositive serum and purified particles was determined. The potential use of HBVinfected hNTCPexpressing PPHs for drug screening was assessed. The results demonstrated that pigs and rabbits are closer to humans in the divergence tree compared with mice and rats, indicating that pigs and rabbits were more likely to facilitate the HBV postentry lifecycle. Following hNTCP complementation and HBV infection, PPHs and Huh7D human hepatocellular carcinoma cells, but not PRHs, exhibited increased hepatitis B surface antigen and hepatitis B eantigen secretion, covalently closed circular DNA formation and infectious particle secretion. hNTCPexpressing PPHs were susceptible to infection with HBV particles purified from pooled HBVpositive sera, but were poisoned by raw HBVpositive sera. The use of HBVinfected hNTCPexpressing PPHs for viral entry inhibitor screening was revealed to be applicable and reproducible. In conclusion, hNTCPexpressing PPHs may be valuable tool for investigating HBV infection and antiviral drugs.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Hepatitis B/drug therapy , Hepatocytes/virology , Organic Anion Transporters, Sodium-Dependent/genetics , Symporters/genetics , Animals , Cells, Cultured , Gene Expression , Hepatitis B/genetics , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Mice , Microbial Sensitivity Tests/methods , Rabbits , Rats , Species Specificity , SwineABSTRACT
BACKGROUND: Accumulating evidence demonstrates that microRNAs (miRNAs) play essential roles in tumorigenesis and cancer progression of hepatocellular carcinoma (HCC). Average targets of a miRNA were more than 100. And one miRNA may act in tumor via regulating several targets. The present study aimed to explore more potential targets of miR-449a by proteomics technology and further uncover the role of miR-449a in HCC tumorigenesis. METHODS: Technologies such as iTRAQ-based quantitative proteomic were used to investigate the effect of miR-449a on HCC. The expression of c-Met and miR-449a was detected by qRT-PCR in HCC samples. Gain- and loss-of-function experiments were performed to identify the function and potential target of miR-449a in HCC cells. RESULTS: In HCC, miR-449a was significantly downregulated, while c-Met was upregulated concurrently. Quantitative proteomics and luciferase reporter assay identified c-Met as a direct target of miR-449a. Moreover, miR-449a inhibited HCC growth not only by targeting CDK6 but also by suppressing c-Met/Ras/Raf/ERK signaling pathway. Furthermore, the inhibition of c-Met expression with a specific siRNA significantly inhibited cells growth and deregulated the ERK pathway in HCC. CONCLUSION: The tumor suppressor miR-449a suppresses HCC tumorigenesis by repressing the c-Met/ERK pathway.
Subject(s)
Carcinoma, Hepatocellular/enzymology , Cell Proliferation , G1 Phase Cell Cycle Checkpoints , Hepatocyte Growth Factor/metabolism , Liver Neoplasms/enzymology , MicroRNAs/metabolism , Proto-Oncogene Proteins c-met/metabolism , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Hepatocyte Growth Factor/genetics , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , Protein Interaction Maps , Proto-Oncogene Proteins c-met/genetics , Signal Transduction , Tumor Cells, CulturedABSTRACT
A method for preparing (103)Pd brachytherapy seeds is reported. The key of the method was to deposit (103)Pd onto carbon bars by electroless plating so as to prepare source cores. After each carbon bar with (103)Pd was sealed in a titanium capsule, the (103)Pd seeds were fabricated. This paper provides valuable experiences and data for the preparation of (103)Pd brachytherapy seeds.