lipofectamine 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

Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine-mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticle (i-LNP)-mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

Topics: Cell Line, Tumor; Gene Expression Profiling; Green Fluorescent Proteins; Humans; Image Processing, Computer-Assisted; Kinetics; Lipids; Liver Neoplasms; Microscopy, Fluorescence; Nanoparticles; Nucleic Acids; Plasmids; RNA, Messenger; Single-Cell Analysis; Transfection

To overcome the rapid enzymatic degradation and low transfection efficiency of siRNA, the delivery carriers for siRNA is a therapeutic demand to increase its stability. Gold nanoparticles (AuNPs) modified by branched polyethyleneimine (bPEI) were developed as an efficient and safe intracellular delivery carriers for siRNA. The current study implied that siRNA designed against an oncogene c-Myc could be delivered by a modified AuNPs complex without significant cytotoxicity. The comparative semi-quantitative and quantitative real time PCR were used to measure the c-Myc gene expression after transfection with naked siRNA and siRNA/bPEI/AuNPs, but AuNPs interfered with PCR. However, the c-Myc protein translation was successfully detected in the transfected HuH7 cells with naked siRNA and siRNA/bPEI/AuNPs and it was found to be inhibited by siRNA/bPEI/AuNPs more than naked siRNA. The results validate the successful silencing of c-Myc gene. Accordingly, it may confirm the promising and effective delivery of siRNA by bPEI/AuNPs. The complex enhances the cellular uptake of siRNA without significant cytotoxicity and confirms that bPEI modified AuNPs could be used as a good candidate for safe cellular delivery of siRNA.

Topics: Cell Line, Tumor; Cell Survival; Citrates; Gene Expression; Gold; Humans; Lipids; Liver Neoplasms; Metal Nanoparticles; Proto-Oncogene Proteins c-myc; RNA, Small Interfering

Lipofectamine 2000 is commonly used for siRNA transfections. However, few studies have examined cellular responses to this delivery system. The purpose of this study is to evaluate the effect of siRNA transfection using Lipofectamine 2000 on cellular autophagy. Huh7.5 cells, stably transfected to express GFP-LC3, were treated with Lipofectamine 2000/negative control siRNA (NC siRNA) complexes. At different time points after treatment, cells were lysed and analyzed by immunoblotting and fluorescence spectroscopy. Cells were also observed using confocal microscopy. An increase of endogenous LC3 lipidation, GFP-LC3 fluorescence, and autophagosomal puncta was observed in cells treated with Lipofectamine 2000/NC siRNA complexes. The kinetics of the increase of GFP-LC3 fluorescence correlated with the concentration of NC siRNA transfected, where 50, 100, and 200 nM NC siRNA caused a significant increase at 72, 48, and 24 h, respectively, after transfection. A similar effect on the GFP-LC3 signal was also observed for cells treated with Lipofectamine 2000 complexed with two other NC siRNAs. The effects were also confirmed in another hepatoma cell line, H4IIE, by immunoblotting. Lipofectamine 2000-mediated transport of NC siRNAs led to an increase of autophagosomes in a dose- and time-dependent manner. Thus, this effect on cells should be taken into consideration when using this approach for intracellular delivery of siRNA.

Topics: Autophagy; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Fluorescence; Green Fluorescent Proteins; Humans; Lipids; Liver Neoplasms; Microtubule-Associated Proteins; Phagosomes; Recombinant Fusion Proteins; RNA, Small Interfering; Transfection

Hepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382-400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.. We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3'UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.. HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3'UTR region, the effect of miR-338-3p on the second binding site (nt 2397-2403) was required for the inhibition.. miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3'UTR region, mainly at nt 2397-2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.

Topics: Blotting, Western; Cell Proliferation; Colony-Forming Units Assay; Cyclin D1; DNA Primers; Flow Cytometry; Hepatitis B virus; Humans; Lipids; Liver Neoplasms; Luciferases; MicroRNAs; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sequence Deletion; Trans-Activators; Viral Regulatory and Accessory Proteins

Gene targeting is currently of distinct interest as an innovative additive treatment option in various malignancies. Its role in pediatric liver tumors has not yet been evaluated thoroughly. For the first time the authors systematically analyzed both lipid-based transfection as well as transduction with adenovirus vectors (Ad) and Sendai virus vectors (SeVV) in order to optimize gene transfer into hepatoblastoma (HB) cells. Two HB cell lines were infected with Ad or SeVV coding for green fluorescent protein (Ad-GFP, SeVV-GFP); transduction efficiencies and apoptosis were assessed using flow cytometry. Furthermore, lipofection of HB cell lines with plasmid-constructs comprising liver-specific promoters was performed using Lipofectamine 2000 and FuGENE 6; lipofection efficiency was monitored by flow cytometry, microscopy, and luciferase activity. The Ad-GFP showed higher transduction rates (61-86%) than the SeVV-GFP (4-24%) depending on the HB cell line used. Infections with first generation SeVV vectors (SeVV-GFP) led to increased target cell apoptosis (7-43%) compared to Ad-GFP (4-16%). The Lipofectamine 2000 revealed a higher transfection efficiency than the FuGENE 6 for both HB cell lines tested. The liver-specific promoters were found to be differently active in the HB cell lines. This study delineates recombinant adenovirus vectors as a promising tool for gene transduction in the HB cells. Furthermore, enhanced activity of the liver-specific promoters in HUH6 cells compared to HepT1 cells supports the observation of varying biological behavior in histologically differing HB tissues.

Topics: Adenoviridae; Cell Line, Tumor; Drug Carriers; Genetic Vectors; Green Fluorescent Proteins; Hepatoblastoma; Humans; Lipids; Liver Neoplasms; Promoter Regions, Genetic; Sendai virus; Transfection

To construct antisense VEGF(165) eukaryotic expression vector PCDNA(3)-as-VEGF(165) and to study its expression and effect on the proliferation of hepatocarcinoma SMMC-7721 cells.. VEGF(165) cDNA was inserted into polylinker sites of eukaryotic expression vector PCDNA(3) to construct PCDNA(3)-as-VEGF(165). Then the vector was transferred into human hepatocarcinoma cell strain SMMC-7721 with cation lipofectamine 2000 mediated methods to evaluate the expression of VEGF protein and the inhibitory effect on the proliferation of hepatocarcinoma SMMC-7721 cells.. The detection indicated the presence of VEGF cDNA in normally cultured SMMC-7721 cells by PCR. VEGF mRNA expression was notably decreased in SMMC-7721 cells by RT-PCR after PCDNA(3)-as-VEGF(165) transfection. The expression of VEGF protein was dramatically inhibited (142.01+/-7.95 vs 1 625.52+/-64.46 pg/ml(-1), P<0.01) 2 days after transfection, which correlated with the dose of PCDNA(3)-as-VEGF(165)5 gene. VEGF protein was most expressed in PCDNA(3) transferred SMMC-7721 cells but few in PCDNA(3)-as-VEGF(165) transferred cells by immunohistochemical staining. The apoptotic rate of hepatocarcinoma SMMC-7721 cells was significantly promoted (17.98+/-0.86% vs 4.86+/-0.27%, P<0.01) and the survival rate was notably decreased (80.99+/-3.20% vs 93.52+/-3.93%, P<0.05) due to antisense VEGF(165) by flow cytometry (FCM). The transfection of antisense VEGF(165) gene resulted in the inhibitory effect on the proliferation of hepatocarcinoma cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and the death of all hepatocarcinoma cells on day 6 after transfection.. It is confirmed that antisense VEGF(165) can inhibit the expression of VEGF protein, interfere with the proliferation and induce the apoptosis of hepatocarcinoma cells in our study. Antisense VEGF(165) gene therapy may play an important role in the treatment of human hepatocarcinoma.

Topics: Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; DNA, Antisense; Humans; Lipids; Liver Neoplasms; Plasmids; Transfection; Vascular Endothelial Growth Factor A

Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA-vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE-mediated transfection in vitro with pcDNA-vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L-02, and RT-PCR was performed afterward. The results of RT-PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA-vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.

Topics: Animals; Apoptosis; Capsid Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Chicken anemia virus; Chickens; Cloning, Molecular; Eukaryotic Cells; Genetic Vectors; Humans; Lipids; Liver Neoplasms; Polymerase Chain Reaction; Recombinant Proteins; Sequence Analysis, DNA; Transfection; Viral Proteins