Recombinant Lentiviral Particles Research Articles

Chicken telomerase reverse transcriptase promotes the tumorigenicity of avian leukosis virus subgroup J by regulating the Wnt/β-catenin signaling pathway

This research aimed to analyze the regulatory effect of chicken telomerase reverse transcriptase (chTERT) on the Wnt/β-catenin signaling pathway and its effect on the tumorigenicity of avian leukosis virus subgroup J (ALV-J) through in vivo experiments. The chTERT eukaryotic expression plasmid and its recombinant lentivirus particles were constructed for in vivo transfection of chTERT to analyze the effect of chTERT continuously overexpressed in chickens on the tumorigenicity of ALV-J. During 156 days of the artificial ALV-J tumor-inducing process, 7 solid tumors developed in 3 chickens in the chTERT-overexpression group (n = 26*2) and no tumors developed in the control group (n = 26*2). Another 18 tumors induced by ALV-J were confirmed and collected from breeding poultry farms. And we confirmed that chTERT was significantly highly expressed in ALV-J tumors. The ELISA data suggested that the protein levels of β-catenin and c-Myc in the chicken plasma of the chTERT-overexpressing group with ALV-J infected were consistently and significantly higher than those of the control group. Compared with that of the tumor-adjacent tissues, the activity of the Wnt/β-catenin signaling pathway and expression of the c-Myc was significantly increased in ALV-J tumors. And the percentage of apoptosis in ALV-J tumors significantly lower than that in tumor-adjacent tissues. Immunohistochemistry, Western blot and RT-qPCR suggested that the replication level of ALV-J in tumors was significantly higher than that in tumor-adjacent tissues. This study suggests that chTERT plays a critical role in the tumorigenicity of ALV-J by enhancing the Wnt/β-catenin signaling pathway, which will contribute to further elucidating the tumor-inducing mechanism of ALV-J.

Effect of WRAP53 β Targeted Co-Inhibitory Pathways Based on Comprehensive Bioinformatics Analysis in Treating Squamous Cell Carcinoma of the Head and Neck

To investigate the association between WD40-encoding RNA antisense to p53 ( WRAP53 β), a telomerase new core subunit, and the clinical, genomic and immune infiltration characteristics of squamous cell carcinoma of the head and neck (HNSC), and to explore for potential joint targeted therapy of HNSC. Tumor IMmune Estimation Resource (TIMER) online modules were adopted to predict the association between WRAP53 β expression and the clinical features, oncogene, and immune infiltration of HNSC in the Cancer Genome Atlas (TCGA) cohort. Tumor Immune Single-cell Hub (TISCH) was used to analyze WRAP53 β expression at the single cell level. Analysis of the small molecule inhibitors potentially targeting WRAP53 β was carried out by Computational Analysis of REsistance (CARE). In the in vitro verification experiment, recombinant lentiviral particles with the sh WRAP53 β sequence were synthesized. Then, the oral squamous cell carcinoma cell line Cal27 (the sh WRAP53 βgroup) stably expressing sh WRAP53 β were constructed, and two control groups were set up (the shNC group consisting of Cal27 cells added with lentiviral particles containing non-specific control sequences and the Con group consisting of untreated Cal27 cells). MTT assay was done to examine the proliferation of cells in the three groups. Cellular immunofluorescence assay was done for further qualitative examination of the expression of P53 protein in the cells of the sh WRAP53 β group and the shNC group. Western blot was done to measure the expression of WRAP53β and γ-H2AX, a DNA damage protein, in the 18 th, 23 rd and 28 th passages of the sh WRAP53 β group and the shNC group. Finally, specimens of 13 cases of oral squamous cell carcinoma and 7 cases of oral mucosal inflammation were collected, and the expression of WRAP53β and γ-H2AX in the clinical specimens of oral squamous cell carcinoma was verified with immunohistochemistry. TIMER analysis revealed that the expression level of WRAP53 β in HNSC tissues was significantly higher than that in normal tissues. There was a significant positive correlation between WRAP53 β expression and multiple genes in the p53 pathway, including CCNB1, CCNB2 and CDK1. Although no significant correlation between WRAP53 β expression and infiltrating immune cells was found, WRAP53 β was significantly positively correlated with the inflammatory factors IFN-γ and IL23A, and negatively correlated with IL-1A and IL-6 in HPV-positive carcinoma of the head and neck. TISCH single cell sequencing datasets also showed higher expression of WRAP53 β in malignant cells, and very low or zero expression in immune cells. According to the CARE scores, the most potent WRAP53 β co-inhibitory drugs were ATM, CDK1 and MDM4 targeted inhibitors. In vitro cell experiments showed that the proliferation ability of Cal27 cells decreased significantly in the sh WRAP53 β group as compared with that of the control group between Day 5 and Day 7 ( P<0.05). Furthermore, the expression of P53 decreased significantly in the sh WRAP53 β group. As compared with the control group, the expression of WRAP53β in sh WRAP53 β group significantly decreased in the 18 th, 23 rd and 28 th passages ( P<0.05), while γ-H2AX expression only decreased in the 18 th and 28 th passages ( P<0.05) according to the results of Western blot. Clinical specimens showed rather high positive expression rate of γ-H2AX in oral squamous cell carcinoma tissues (12/13), while the expression of WRAP53β was not detected in oral mucositis samples (0/7). WRAP53 β showed significantly higher expression level in HSNC, and was significantly associated with p53 pathway genes. ATM, CDK1 and MDM4 inhibitors may be potential WRAP53 β co-inhibitory agents. RNA interference of WRAP53 β expression may cause inhibition of DNA damage, thereby indicating therapeutic potential for HNSC.

Experimental Study of the In Vitro and In Vivo Functional Activity of NKG2D Chimeric Antigen Receptor

Aim. To study antitumor cytotoxic effect of CAR-T NKG2D and CAR-T anti-CD19 in vitro and in vivo in order to compare antitumor activity of chimeric antigen receptors (CAR) with different structural and functional properties. Materials &amp; Methods. CAR constructions were produced by molecular cloning. CAR-T cell populations were obtained by transduction of healthy donor T-lymphocytes with recombinant lentiviral particles coding CAR NKG2D or CD19 target antigen CAR sequences. CAR-T cell proportion was assessed by FusionRed fluorescence and EGFR membrane receptor imaging. Specific in vitro cytotoxic activity of CAR-T effector cells was analyzed by Real-Time Cytotoxicity Assay (RTCA) during co-cultivation with HeLa_CD19 target cell line using xCELLigence. Interferon-Y (IFN-y) synthesis in vitro and in vivo along with the degree of cytotoxic effect were analyzed by immunoassay of culture medium of co-cultivated effector cells and target cells as well as isolated auto-plasma from the peripheral blood of mice. To assess the in vivo functional activity, CAR-T cell populations were infused into immunodeficient NSG-SGM3 mice (10 000 000 cells/mouse) 12 days after HeLa_CD19 cell injection and confirmation of engraftment and tumor growth. Upon euthanasia, tumors were removed and fixed in paraffin to prepare histological sections. CAR-T cell tumor infiltration was assessed by CD3 antigen immunohistochemical staining. Results. The highest ligand (molecules MICA, ULBP1/2/3/4/5/6) expression levels were detected in HeLa cell line. The obtained NKG2D CAR-T cells showed a considerable cytotoxic activity against HeLa_CD19 target line (cell index [CI] = 1.27), which was, however, twice as low as that of CAR-T anti-CD19 (CI = 0.60) (p = 0.0038). IFN-y level during co-cultivation of CAR-T anti-CD19 with HeLa_CD19 at the ratio of Е/Т = 1:1 was 64,852 pcg/mL, which was 3.5 times higher than IFN-y level during co-cultivation of CAR-T NKG2D with HeLa_CD19 (18,635 pcg/mL) (p = 0.0360). The degree of tumor infiltration by CAR-T anti-CD19 cells was higher than that by CAR-T NKG2D. The absence of NKG2D proliferating CAR-T cells in mice peripheral blood confirms their low persistence. IFN-y concentration in mice auto-plasma was 11.89 pcg/mL after CAR-T anti-CD19 infusion and 0.57 pcg/mL after CAR-T NKG2D infusion (p = 0.0079). The mean weight of tumor xenografts in experimental groups 10 days after CAR-T anti-CD19 injection was 0.72 g (p = 0.0142), after Т-lymphocyte and NKG2D CAR-T cell infusions it was 2.12 g and 1.2 g, respectively. Conclusion. CAR-T anti-CD19 cells are characterized by more pronounced cytotoxic effect under both in vitro and in vivo experimental conditions compared with CAR-T NKG2D cells. The degree of CAR-T anti-CD19 proliferation and their infiltration in mice xenograft models is considerably higher than the levels reached with NKG2D CAR-T cell injections. A single CAR-T NKG2D injection results only in short-term tumor reduction.

Overexpression Effects of miR-424 and BMP2 on the Osteogenesis of Wharton's Jelly-Derived Stem Cells.

Recently, the translational application of noncoding RNAs is accelerated dramatically. In this regard, discovering therapeutic roles of microRNAs by developing synthetic RNA and vector-based RNA is attracting attention. Here, we studied the effect of BMP2 and miR-424 on the osteogenesis of Wharton's jelly-derived stem cells (WJSCs). For this purpose, human BMP2 and miR-424 DNA codes were cloned in the third generation of lentiviral vectors and then used for HEK-293T cell transfection. Lentiviral plasmids contained miR424, BMP-2, miR424-BMP2, green fluorescent protein (GFP) genes, and helper vectors. The recombinant lentiviral particles transduced the WJSCs, and the osteogenesis was evaluated by real-time PCR, Western blot, Alizarin Red staining, and alkaline phosphatase enzyme activity. According to the results, there was a significant increase in the expression of the BMP2 gene and secretion of Osteocalcin protein in the group of miR424-BMP2. Moreover, the amount of dye deposition in Alizarin Red staining and alkaline phosphatase activity was significantly higher in the mentioned group (p < 0.05). Thus, the current study results clarify the efficacy of gene therapy by miR424-BMP2 vectors for bone tissue engineering. These data could help guide the development of gene therapy-based protocols for bone tissue engineering.

Phosphorylation of CREB-Specific Coactivator CRTC2 at Ser238 Promotes Proliferation, Migration, and Invasion of Colorectal Cancer Cells.

cAMP response element binding protein (CREB)-regulated transcription coactivator 2 (CRTC2), a member of the novel CRTC family of transcriptional coactivators that activates basic leucine zipper transcription factors, including CREB, is overexpressed in many carcinomas, including colon cancer. Phosphorylation of CRTC2 protein at different residues is important for its subcellular localization and activity. However, the functions of some of the serine phosphorylation sites have not been elucidated. This study aimed to investigate the effects of phosphorylation of Ser127, Ser238, and Ser245 sites of CRTC2 in colorectal cancer (CRC) cells. Recombinant lentiviral particles with a CRTC2-targeting small hairpin RNA (shRNA) sequence were transfected into CRC cells to obtained shCRTC2 cell lines. Site-directed mutagenesis of Ser127, Ser238, and Ser245 cells were constructed by transfecting CRTC2 cDNA containing S127A, S238A, and S245A mutations into shCRTC2. Cell proliferation was measured by cell counting kit-8. Cell migration and invasion were examined by transwell assay. mRNA expression was assayed by qRT-PCR, and protein expression was determined by Western blot. Our results indicate that CRTC2 is overexpressed in CRC cells. Knockdown of CRTC2 inhibits the proliferation, migration, and invasion of CRC cells. When the phosphorylation of CRTC2 Ser238 decreases due to the lack of ERK2, the phosphorylation of Ser171 site increases. The proliferation, migration and invasion of CRC cells were inhibited, the nuclear aggregation of CRTC2 in the nucleus was reduced, and the interaction between CRTC2 and CREB was weaken. It is shown that the phosphorylation of CRTC2 Ser238 is important for CREB transcriptional activity. These findings may help in the identification of potentially new targets for CRC therapy.

Hepatitis C virus nonstructural protein 5A perturbs lipid metabolism by modulating AMPK/SREBP-1c signaling

BackgroundSteatosis is an important clinical manifestation associated with chronic hepatitis C virus (HCV) infection. AMP-activated protein kinase (AMPK), a major mediator of lipid metabolism, regulates HCV-associated hepatic steatosis, but the underlying mechanisms remain obscure. Here we investigated the mechanism of HCV nonstructural protein 5A (NS5A)-induced lipid accumulation by the AMPK/SREBP-1c pathway.MethodsWe generated model mice by injecting recombinant lentiviral particles expressing the NS5A protein (genotype 3a) via the tail vein. The serum levels of alanine aminotransferase (ALT), free fatty acids (FFAs) and triglycerides (TG) were examined. H&E and Oil Red O staining were used to examine lipid droplets. Immunohistochemistry staining, quantitative real-time PCR and Western blotting were used to determine the expression of lipogenic genes.ResultsOur results showed that the serum levels of ALT, FFAs and TG, as well as the accumulation of hepatic lipid droplets, were increased significantly in mice infected with NS5A-expressing lentiviral particles. NS5A inhibited AMPK phosphorylation and increased the expression levels of sterol regulatory element binding protein-1c (SREBP-1c), acetyl-coenzyme A carboxylase 1 (ACC1) and fatty acid synthase (FASN) in vivo and in vitro. Further investigation revealed that pharmacological activation or ectopic expression of AMPK neutralized the upregulation of SREBP-1c, ACC1 and FASN, and ameliorated hepatic lipid accumulation induced by NS5A. Ectopic expression of SREBP-1c enhanced NS5A-induced hepatic lipid accumulation, which was dramatically reversed by pharmacological activation of AMPK.ConclusionsCollectively, we demonstrate that NS5A induces hepatic lipid accumulation via the AMPK/SREBP-1c pathway.

A gene-based anti-angiogenesis therapy as a novel strategy for cancer treatment

Angiogenesis-targeted therapy of cancer is considered a promising strategy for therapeutic management of cancer progression. Over the last two decades, a few anti-angiogenesis monoclonal antibodies (mAbs) blocking VEGF signaling have been developed and approved by the FDA. The most widely used anti-angiogenesis drug is bevacizumab which binds VEGFA and prevents its interaction with VEGF receptor leading to suppression of angiogenesis. Despite the remarkable success in development of angiogenesis inhibitory mAbs, their clinical application is limited by the high-cost of mAbs-based regimen which includes multiple doses of mAbs due to their short biological half-life. Antibody gene therapy is an alternative system of antibody production. In this study, we have developed a gene-based anti-VEGF mAb system which is expected to produce a high concentration of anti-VEGFA mAb upon a single administration in cancer patients. The full-length cDNA bevacizumab light and heavy chains joint with T2A sequence were cloned in pCDH lentivirus vector. The lentiviral particles expressing bevacizumab was produced in HEK-293T cells. Recombinant lentiviral particles containing bevacizumab (rLV-bev) efficiently transduced HEK-293cells and produced functional bevacizumab mAb. Bevacizumab expression in the transduced cell was assessed by qRT-PCR and western blot at both the mRNA and protein level, respectively. The functionality of the recombinant bevacizumab was confirmed using the tube formation assay in the co-culture system of endothelial cells and HT-29cells transduced with rLV-bev viral particles. Our results show that rLV-bev gene therapy can be useful for angiogenesis-targeted therapy of cancer.

Daxx overexpression inhibits AngⅡ-induced proliferation and migration in vascular smooth muscle cells

To construct a recombinant lentiviral expression vector pCDH-Daxx-EGFP to investigate the effect of Daxx on the proliferation of vascular smooth muscle cells (VSMCs). The recombinant lentiviral expression vector pCDHDaxx-EGFP was constructed using PCR-based accurate synthesis method. After identification by sequencing and enzyme digestion, the recombinant lentiviral vector was contransfected into 293T cells with lentivirus packaging vector. The recombinant lentivirus particles were collected and purified to infect VSMCs, whose expression of Daxx was detected with Western boltting. The cells infected with the empty vector pCDH-EGFP or pCDH-Daxx-EGFP were incubated in serum-free medium or in the presence of angiotensin Ⅱ (AngⅡ). The cell viability was determined with MTT assay, and the cell cycle changes were analyzed with flow cytometry. The cell migration ability was assessed using a scratch wound healing assay. The expression of p-Akt protein in the cells was detected using Western blotting. Double enzyme digestion and sequencing confirmed successful construction of the recombinant plasmid. Compared with the cells infected with the empty vector, the cells infected with pCDH-Daxx-EGFP exhibited significantly increased expressions of Daxx protein (P < 0.05). AngⅡ treatment of the cells infected with the pCDH-Daxx-EGFP, as compared with the cells infected with the empty vector, significantly lowered the cell viability, S phase cell ratio and cell migration ability (P < 0.05), and significantly decreased the expression level of p-Akt protein (P < 0.05). We successfully constructed the recombinant lentiviral vector pCDH-Daxx-EGFP and overexpressed Daxx in primary cultured VSMCs using this vector. Daxx overexpression can inhibit AngⅡ-induced proliferation and migration in VSMCs probably by regulating p-Akt protein.

Expression and function of long non-coding RNA epigenetically-induced 1 in hepatocellular carcinoma

Objective To observe the expression of long non-coding RNA (lncRNA EPIC1) in hepatocellular carcinoma (HCC) and its effects on the proliferation, invasion and apoptosis of hepatoma cell lines. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was adopted to detect the expression levels of EPIC1 in 7 cases of HCC tissues and their paracancerous tissues, 5 liver cancer cell lines (HepG2, SK-HEP-1, HuH-7-80, SMMC-7721, SNU-387) and normal human liver cell line (HL-7702). Hep G2 and HuH-7-80 cells were transfected with recombinant lentiviral particles carrying the EPIC1 genes [LV-small interfering RNA (siRNA)] as the experimental group, and Hep G2 and HuH-7-80 cells transfected with negative control lentiviral particles (LV-NC) were used as the control group. The proliferative capacity of HepG2 and HuH-7-80 cells was measured by cell counting kit-8 (CCK-8) proliferation assay after transfection. The invasion capacity of HepG2 and HuH-7-80 cells was examined by Transwell invasion assay. The apoptosis rate of HepG2 and HuH-7-80 cells was detected by flow cytometry. Results The expression level of EPIC1 in HCC tissues (8.77±0.36) was significantly higher than that in their adjacent tissues (2.39±0.45), and the difference was statistically significant (P<0.01). The expression levels of EPIC1 in HepG2, SK-HEP-1, HuH-7-80, SMMC-7721 and SNU-387 cells (7.24±0.81, 3.11±0.35, 5.17±1.12, 4.38±0.48 and 2.69±0.65) was significantly [about (5.61±0.12) and (4.01±0.16) times] higher than that of HL-7702 cells (1.29±0.13), among which HepG2 and HuH-7-80 cells had the highest expression level, and the difference was statistically significant (P<0.01). The proliferative capacity of HepG2 cells in the EPIC1 group with low expression at 24, 48, 72 h (LV-siRNA1: 0.43±0.02, 0.72±0.02, 1.31±0.01, P<0.05; LV-siRNA2: 0.42±0.02, 0.76±0.04, 1.03±0.02, P<0.01) was significantly lower than HepG2 cells (0.59±0.02, 0.98±0.02, 1.63±0.02) in the control group. The proliferative capacity of HuH-7-80 cells with low expression in the EPIC1 group at 24, 48, 72 h (LV-siRNA1: 0.59±0.01, 0.73±0.02 and 1.31±0.05, P<0.05; LV-siRNA2: 0.43±0.04, 0.74±0.04 and 1.26±0.04, P<0.01) was significantly lower than that of the HuH-7-80 cells (0.64±0.02, 0.82±0.02 and 1.78±0.02) in the control group. The number of cells across the basement membrane in the EPIC1 group with low expression in HepG2 and HuH-7-80 cells (43.35±6.16, 55.40±8.18) was significantly less than that in the control group (80.67±5.14 and 74.73±6.13) (P<0.05). The apoptosis rate of HepG2 and HuH-7-80 cells with low expression in EPIC1 group was significantly higher than that in the control group [(2.60±0.96)% and (3.60±0.33)%], and the difference was statistically significant (P<0.01). Conclusion lncRNA EPIC1 is highly expressed in HCC tissues and cells. EPIC1 with low expression can reduce the capacity of proliferation and invasion of hepatoma cells and promote the apoptosis of hepatoma cells. Key words: Carcinoma, hepatocellular; Long non-coding RNA EPIC1; Proliferation; Invasion; Apoptosis

Artificial MicroRNA-Mediated Tgfbr2 and Pdgfrb Co-Silencing Ameliorates Carbon Tetrachloride-Induced Hepatic Fibrosis in Mice.

Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrogenesis. Transforming growth factor beta 1 (TGF-β1) and platelet-derived growth factor (PDGF) are key profibrotic cytokines that regulate HSC activation and proliferation with functional convergence. Dual RNA interference against their receptors may achieve therapeutic effects. A novel RNAi strategy based on HSC-specific GFAP promoter-driven and lentiviral-expressed artificial microRNAs (amiRNAs) was devised that consists of an microRNA-30a backbone and effective shRNAs against mouse Pdgfrβ and Tgfbr2. Then, its antifibrotic efficacy was tested in primary and cultured HSCs and in mice affected with carbon tetrachloride-induced hepatic fibrosis. The study shows that amiRNA-mediated Pdgfrβ and Tgfbr2 co-silencing inhibits HSC activation and proliferation. After recombinant lentiviral particles were delivered into the liver via tail-vein injection, therapeutic amiRNAs were preferentially expressed in HSCs and efficiently co-knocked down in situ Tgfbr2 and Pdgfrβ expression, which correlates with downregulated expression of target or effector genes of their signaling, which include Pai-1, P70S6K, and D-cyclins. amiRNA-based HSC-specific co-silencing of Tgfbr2 and Pdgfrβ significantly suppressed hepatic expression of fibrotic markers α-Sma and Col1a1, extracellular matrix regulators Mmps and Timp1, and phenotypically ameliorated liver fibrosis, as indicated by reductions in serum alanine aminotransferase activity, collagen deposition, and α-Sma-positive staining. The findings provide proof of concept for the use of amiRNA-mediated co-silencing of two profibrogenic pathways in liver fibrosis treatment and highlight the therapeutic potential of concatenated amiRNAs for gene therapy.

Construction of human ARF4 lentiviral vector and stable expression in ovarian cancer cell line SKOV3

Objective To establish ovarian cancer cell line SKVO3 that can stably express human ADP ribosylation factor-4 (ARF4). Methods A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was constructed and transfected into SKOV3 cells after verifying by DNA sequencing. The expression of ARF4 mRNA was verified by real-time quantitative PCR (qRT-PCR). Then, the recombinant plasmid with lentiviral packaging plasmids were co-transfected into SKOV3 cells for packaging. The recombinant lentiviral particles LV-ARF4 were collected and transfected into SKOV3 cells, and the stable transfected SKOV3 cell line was screening by culture with puromycin. The expression of ARF4 gene was detected by qRT-PCR and Western Blot. Results A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was successfully constructed. The vector could significantly up-regulate the expression of ARF4 mRNA in SKOV3 cells and be successfully packaged into recombinant lentiviral particles in HEK-293T cells. Compared with the control group, the relative expression level of ARF4 mRNA and protein in SKOV3 cells was significantly increased after the infection with LV-ARF4 (all P<0.001). Conclusion The recombinant plasmid pCDH-CMV-MCS-EF1-Puro/ARF4 and lentiviral vector LV-ARF4 were successfully constructed. The establishment of stably infected SKOV3 cell line with LV-ARF4 provides an experimental foundation for further studies on the biological function of ARF4 in ovarian cancer. Key words: Gene cloning; Lentiviral vector; ADP-ribosylation factor 4; SKOV3 cell line

Effect of hepatitis C virus nonstructural protein 5A and its domains II on hepatocyte gluconeogenesis in mice

Objective: To investigate the role of hepatitis C virus nonstructural protein 5A (NS5A) and its domains I, II, and III in regulating gluconeogenesis in mice and the underlying mechanism. Methods: A total of 60 male C57BL/6J mice were randomly divided into six groups. Recombinant lentiviral particles with specific expression of full-length NS5A, NS5A domain I, NS5A domain II, or NS5A domain III were injected via the caudal vein to establish a mouse model, and the group without injection and the group with the injection of the lentiviral particles containing enhanced green fluorescent protein (EGFP) were established as negative control. The effect of full-length NS5A protein and its domains on fasting blood glucose (FBG) and fasting serum insulin (FINS) were measured. Liver tissue was collected to prepare a paraffin section. Immunohistochemistry was used to measure the expression of phosphoenolpyruvate carboxykinase (PEPCK) in hepatocytes, quantitative real-time PCR and/or Western blot were used to measure the expression of NS5A, phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), sterol regulatory element-binding protein-1 (SREBP-1), and PEPCK. Results: Compared with the group without injection and the group with the injection of the lentiviral particles containing EGFP, the groups with the injection of the lentiviral particles containing full-length NS5A and NS5A domain II had significant increases in FBG and homeostasis model assessment of insulin resistance index (P < 0.01). Immunohistochemistry and quantitative real-time PCR showed a significant increase in the expression of PEPCK, a key enzyme involved in gluconeogenesis. Western blot showed that full-length NS5A protein and NS5A domain II inhibited the level of p-AMPK and increased the levels of SREBP-1 and PEPCK. Conclusion: NS5A protein and NS5A domain II may affect glucose metabolism in hepatocytes in mice by regulating AMPK/SREBP-1/PEPCK, and NS5A domain II may play an important role in insulin resistance in hepatocytes caused by HCV infection.

Construction and Evaluation of MKN45 DEC1 Knockout Gene Stable Strain by Using CRISPR/Cas9 Gene Editing System

Human differentiated embryonic chondrocyte-expressed gene 1 (DEC1) (also known as Stra13/Bhlhb2/Sharp2) is a basic helix-loop-helix (bHLH) transcription factor. Our previously studies have shown that the upregulation of DEC1 play an important role in cell proliferation in gastric cancer (GC). The latest gene editing system, CRISPR/Cas9 technology uses sequence-specific RNA molecules guide endonuclease Cas9 to the target nucleic acid and active the endonuclease to cut the double-stranded DNA, thereby complete genome knockout. Therefore, we use the CRISPR/ Cas9 system to completely knockout DEC1 genes in GC. Two gRNAs against DEC1 were designed. 293T cells were transfected using Lipofectamine 2000 and packaged for the recombinant lentivirus particles. Subsequently, we collected the lentivirus venom to infect the MKN45 cells and establish a stable, knockout cell line named MKN45-DEC1-KO. This study will provide a new cell model for further study of the role of DEC1 in the pathogenesis of gastric cancer.

P08.69 A lentiviral-based system for targeting the regulatory subunit R2A of protein kinase A in human glioblastoma

Introduction: Glioblastoma (GBM) is the most frequent and lethal primary brain tumor, associated with a median survival of 12-15 months after first diagnosis. The identification of new therapeutic approaches is required for the development of effective treatments. The cAMP-activated protein kinase (PKA) pathway is dysregulated in many pathological conditions, including cancer, and could potentially provide new therapeutic targets for GBM treatment. In rodent and human GBM cells the regulatory subunit R2A of PKA presents a specific localization on Golgi apparatus, that is not detectable in the healthy brain parenchyma. Moreover, downregulation of PKA R2A expression induces a reduction of GBM cell viability. In order to deeply investigate the effects of stable PKA R2A downregulation, a lentiviral-based system was developed for delivery of short hairpin RNA (shRNA) targeting PKA R2A gene to human GBM cells. The 3rd generation lentiviral vector cPPT.hCMV.EGFP was first tested for transduction of U87 human GBM cell line and primary GBM cell cultures. Then, recombinant lentiviral vectors were generated for expression of shRNA targeting PKA R2A gene. This lentiviral system represents an efficient strategy for shRNA delivery and downregulation of PKA R2A expression. Material and method: U87 human GBM cell line and primary GBM cell cultures were grown respectively as adherent cells in serum-supplemented medium and as spheres in serum-free medium. 293T cells were used for production and titration of lentiviral vectors. EGFP expression was monitored at epifluorescence microscope and western blot analysis was performed to evaluate R2A expression. Results and discussion: The lentiviral cPPT.hCMV.EGFP vector is able to efficiently transduce U87 cells and primary GBM spheres. Transduced U87 cells maintain the expression of the EGFP reporter gene for more than two weeks and no apparent influence on cell growth was observed. Expression of EGFP is detected from most, but not all, cells forming the transduced primary GBM spheres. Moreover, transduced primary GBM cells are able to form spheres and maintain the expression of the reporter gene after dissociation. Recombinant lentiviral cPPT.hCMV.EGFP-based vectors were generated for expression of shRNA.Temp1 or shRNA.Temp2, targeting different exons of PKA R2A gene, and for their co-expression with EGFP. Western blot analysis demonstrated an efficient reduction of R2A expression in cells transfected with the cis-elements of recombinant vectors and in cells transduced with the recombinant lentiviral particles. Conclusion: The 3rd generation lentiviral cPPT.hCMV.EGFP vector proved to efficiently transduce human GBM cells and primary GBM spheres. This lentiviral system allows efficient delivery of shRNA for downregulation of PKA R2A expression, thus representing a new strategy for targeting protein kinase A in human GBM cells.

Reduced RKIP expression levels are associated with frequent non-small cell lung cancer metastasis and STAT3 phosphorylation and activation.

The current study examined the role of Raf kinase inhibitor protein (RKIP) in non-small cell lung cancer (NSCLC) metastasis. A total of 100 patients with NSCLC were recruited following pathological diagnosis in the First Affiliated Hospital of Bengbu Medical College. The patients were classified and statistically analyzed according to their clinicopathological characteristics and tumor-node-metastasis stage. Paired tumor tissue and adjacent non-tumor tissue samples were subject to pathological diagnosis and western blot analysis. Transient transfection and lentivirus particle vector-mediated RKIP overexpression, small interfering RNA-mediated silencing, Transwell assays and immunocytochemistry methods were employed to elucidate the role and underlying mechanisms of RKIP and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway in NSCLC metastasis. Furthermore, in order to examine the in vivo effects of RKIP, recombinant lentivirus particles containing the RKIP gene were administrated in a mouse NSCLC tumor model via tail vein injection. The results revealed reduced RKIP expression levels in NSCLC tissue compared with corresponding non-cancer tissue. Additionally, RKIP expression levels were inversely associated with NSCLC intra-lung, lymph node and long-distance metastasis. The results also indicated that RKIP was able to block STAT3 activation via phosphorylation and inhibit NSCLC-cell metastasis in vitro. Furthermore, RKIP knockdown was able to promote STAT3 phosphorylation and cell metastasis in NSCLC cell lines. During in vivo experiments, RKIP overexpression was able to suppress xenograft tumor metastasis in nude mice. Therefore, RKIP may be an important factor in cancer cell metastasis in patients with NSCLC, and RKIP may inhibit NSCLC-cell invasion by blocking the activation of the JAK/STAT3 signaling pathway.

Small RNAs targeting the 5′ end of the viral polymerase gene segments specifically interfere with influenza type A virus replication

Human and avian influenza A viruses, associated with seasonal epidemics and occasionally with pandemics, have a high impact on public health. The development of new antivirals to counteract the emergence of drug resistant influenza virus variants is a main concern. The aim of this study was to develop systems for the efficient and stable expression of small therapeutic RNAs into influenza virus infected cells in order to get further insights on the efficacy of nucleic acid-based antiviral strategies. To this end, lentiviral vectors expressing either microRNAs or antisense-RNAs targeting the 5′ end of the PA, PB1 and PB2 influenza virus genomic sequences were generated. Derivative recombinant lentiviral particles were employed to transduce the influenza virus highly susceptible human alveolar basal epithelial A549 cells. The expression of both RNA molecules led to a reduction up to 3 logs of the viral titer when transduced A549 cells were challenged with different human and avian subtypes of influenza type A virus. Importantly, no inhibition of influenza type B virus was observed. Overall our data support the development of nucleic acid-based antiviral strategies to control human and avian influenza A virus infection.

MiRNA-155 modulates the malignant biological characteristics of NK/T-cell lymphoma cells by targeting FOXO3a gene.

This study investigated the effects of miRNA-155 on malignant biological characteristics of NK/T-cell lymphoma cell lines and the possible mechanism. The expression of miRNA-155 was detected in lymphoma cell lines from different sources (SNK-6, YTS, Jurkat and DOHH2) by real-time PCR. Lentiviral vectors (pLL3.7) that could overexpress or downexpress miRNA-155 were constructed. Recombinant lentiviral particles were prepared and purified, and their titers determined. The expression of miRNA-155 in the infected SNK-6 cells and the cell proliferation were detected by PCR and CCK-8, respectively. Flow cytometry was used to determine the apoptosis of infected SNK-6 cells. The target of miRNA155 was predicted from Targetscan website. The effect of miRNA155 on FOXO3a expression was examined by Western blotting. The results showed that among the human NK/T-cell lymphoma cell lines SNK-6, YTS, Jurkat and DOHH2, the expression of miRNA-155 was highest in SNK-6. The infection efficiency of the recombinant lentivirus in SNK-6 was more than 70% at multiplicity of infection (MOI) of 100. The expression of miRNA-155 was significantly increased in SNK-6 cells infected by lentivirus vectors with high expression of miRNA-155 (4 times higher than the control group), and profoundly decreased in those infected with lentiviruses with low expression of miRNA-155. The proliferation of letivirus-infected SNK-6 cells was decreased as the expression of miRNA-155 reduced. The apoptosis rate was increased with the reduction in the expression of miRNA-155. FOXO3a was found to be a possible target of miRNA155, as suggested by Targetscan website. Western blotting showed that the expression of FOXO3a was significantly elevated in SNK-6 cells with miRNA-155 inhibition. It was concluded that reduction in miRNA-155 expression can inhibit the proliferation of SNK-6 lymphoma cells and promote their apoptosis, which may be associated with regulation of FOXO3a gene.

A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications.

Effect of overexpression of CAV1 mediated by lentivirus on proliferation and apoptosis of HL-60 cells

This study was purposed to explore the effect of lentivirus-mediated CAV1 overexpression on proliferation and apoptosis in HL-60 cells. Recombinant lentiviral expression vector pcDNA-EF1-CAV1 was constructed, and cotransfected the 293TN cells with a mixture of pPACK packaging plasmids. Then collecting virus suspension infects the HL-60 cells, which make CAV1 gene stable transfection and high expression in the cells. The CAV1 protein expression status in HL-60 cells transfected was evaluated through Western blot method. Proliferative activity and apoptosis of HL-60 cells before and after transfection were detected by CCK-8 method and flow cytometry, respectively. The results showed that the PCR-positive clone screening and results of nucleotide sequencing confirmed that the CAV1 gene inserted into the expression vector pcDNA-EF1-GFP correctly, recombinant lentiviral particles Lv-CAV1 transfected HL-60 cells successfully and with transfection rate up to 90%. The result of Western blot showed that CAV1 protein expression in HL-60 cells significantly increased at 48 hours after transfection. CCK-8 result indicated that cell proliferation activity increased at 48 h after transfection (P < 0.05), flow cytometry testing results displayed that apoptosis rate of HL-60 cells obviously decreased after transfection (P < 0.01). It is concluded that the overexpression of CAV1 in HL-60 cells can inhibit cell proliferation activity and promote cell apoptosis.

Production of transgenic calves expressing an shRNA targeting myostatin

Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing.