cyanine-dye-3 and Liver-Neoplasms

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.

Topics: Base Sequence; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Fluorescent Dyes; Gene Silencing; Gene Transfer Techniques; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Lipid Metabolism; Lipids; Liposomes; Liver Neoplasms; Polymers; RNA Interference; RNA, Small Interfering; Transfection

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

Topics: Aged; alpha-Fetoproteins; Antibodies; Area Under Curve; Biomarkers, Tumor; Carbocyanines; Carcinoma, Hepatocellular; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Fluorescence; Fluorescent Dyes; Gene Expression; Humans; Liver Neoplasms; Male; Middle Aged; Protein Array Analysis; ROC Curve; Streptavidin

The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A.

Topics: Actin Cytoskeleton; Antibodies, Monoclonal; Carbocyanines; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Colchicine; Cytochalasin B; Fluorescein-5-isothiocyanate; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Hepacivirus; Humans; Indoles; Kidney; Liver Neoplasms; Microtubules; Replicon; RNA Helicases; RNA, Viral; Serine Endopeptidases; Tubulin; Tubulin Modulators; Viral Nonstructural Proteins; Virus Replication

Hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). HBV encodes the potentially oncogenic HBx protein, which mainly functions as a transcriptional co-activator involving in multiple gene deregulations. However, mechanisms underlying HBx-mediated oncogenicity remain unclear. To determine the role(s) of HBx in the early genesis of HCC, we utilized the NCI Oncochip microarray that contains 2208 human cDNA clones to examine the gene expression profiles in either freshly isolated normal primary adult human hepatocytes (Hhep) or an HCC cell line (SK-Hep-1) ecotopically expressing HBx via an adenoviral system. The gene expression profiles also were determined in liver samples from HBV-infected chronic active hepatitis patients when compared with normal liver samples. The microarray results were validated through Northern blot analysis of the expression of selected genes. Using reciprocally labeling hybridizations, scatterplot analysis of gene expression ratios in human primary hepatocytes expressing HBx demonstrates that microarrays are highly reproducible. The comparison of gene expression profiles between HBx-expressing primary hepatocytes and HBV-infected liver samples shows a consistent alteration of many cellular genes including a subset of oncogenes (such as c-myc and c-myb) and tumor suppressor genes (such as APC, p53, WAF1 and WT1). Furthermore, clustering algorithm analysis showed distinctive gene expression profiles in Hhep and SK-Hep-1 cells. Our findings are consistent with the hypothesis that the deregulation of cellular genes by oncogenic HBx may be an early event that favors hepatocyte proliferation during liver carcinogenesis.

Topics: Adult; Blotting, Northern; Carbocyanines; Carcinoma, Hepatocellular; Fluorescent Dyes; Freezing; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Humans; Liver; Liver Neoplasms; Oligonucleotide Array Sequence Analysis; Staining and Labeling; Trans-Activators; Tumor Cells, Cultured; Viral Regulatory and Accessory Proteins