Search

Target gene silencing is a strategy that can be used to turn off pathways or genes which are difficult to turn off pharmacologically, both because of lack of targeting drugs, or because of the risk of wider off-target effects. Here we describe the design and use of short hairpin RNA (ShRNA) and lentiviral vectors as an efficient technique for silencing NF-kappaB (NF-κB) pathway in cultured cells. This method can be used also in hard to transfect primary cell cultures.

Remodeling of the gene regulatory network in cells is believed to be a prerequisite for their lineage reprogramming. However, its key regulatory factors are not yet elucidated. In this article, we investigate the role of PIWI proteins and provide evidence that one of them, MIWI2, is elicited during transdifferentiation of fibroblasts into hepatocyte-like cells. In coincidence with the peak expression of MIWI2, we identified the appearance of a unique intermediate epigenetic state characterized by a specific Piwi-interacting RNA (piRNA) profile consisting of 219 novel sequences. Knockout of MIWI2 greatly improved the formation of the induced hepatocytes, whereas overexpression of exogenous MIWI2 completely abolished the stimulated effect. A bioinformatics analysis of piRNA interaction network, followed by experimental validation, revealed the Notch signaling pathway as one of the immediate effectors of MIWI2. Altogether, our results show for the first time that temporal expression of MIWI2 contributes negatively to cell plasticity not only in germline, but also in developed cells, such as mouse fibroblasts. Stem Cells 2019;37:803-812., (©2019 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2019.)

Systemic inflammatory response syndrome is a pathophysiological inflammatory response mediated largely by tumor necrosis factor‑α (TNF‑α), in response to infectious or non‑infectious stimuli. TNF‑α secretion in response to bacterial lipopolysaccharide (LPS) is regulated in part by disintegrin and metalloproteinase domain‑containing protein 17 (ADAM17). Therefore, the present study aimed to identify an effective inhibitor of ADAM17, in order to control inflammation and associated processes. In the present study, a lentiviral vector expressing short hairpin (sh)RNA targeting the ADAM17 gene was constructed. U937 cells were infected with the lentivirus and stimulated with LPS. ADAM17 expression was assessed by western blotting and TNF‑α secretion was assessed by ELISA analysis. The lentivirus was additionally tested in vivo in a mouse model of endotoxemia and sTNF‑α expression was assessed by flow cytometry in peritoneal macrophages. In vitro, the ADAM17 shRNA lentivirus reduced ADAM17 expression, and prevented TNF‑α maturation in U937 cells. In vivo, mice exposed to the ADAM17 shRNA lentivirus prior to LPS‑induced endotoxemia exhibited fewer signs of inflammation and less tissue damage compared with the control mice. In conclusion, the present study successfully constructed a shRNA lentiviral vector targeting the ADAM17 gene that exhibited apparent in vitro and in vivo effects on TNF‑α processing in response to an LPS challenge. The results of the present study may aid the design and improvement of drugs designed to inhibit the function of ADAM17, and suggested a novel means of controlling inflammation and associated processes.

RNA silencing is used as a common method for investigating loss-of-function effects of genes of interest. In mammalian cells, RNA interference (RNAi) or RNA silencing can be achieved by transient siRNA (small or short interfering RNA) transfection or by stable shRNA (short hairpin RNA) systems. Various vectors are used for efficient delivery of shRNA. Lentiviral vectors offer an efficient delivery system for stable and long-term expression of the shRNA in mammalian cells. The widely used lentiviral pLKO.1 plasmid vector is very popular in RNAi studies. A large RNAi database, a TRC (the RNAi Consortium) library, was established based on the pLKO.1-TRC plasmid vector. This plasmid (also called pLKO.1-puro) has a puromycin-resistant gene for selection in mammalian cells along with designs for generating lentiviral particles as well for RNA silencing. While using the pLKO.1-puro TRC control shRNA plasmid for transfection in murine P19 embryonic stem (ES) cells, it was unexpectedly discovered that this plasmid vector induced robust endodermal differentiation. Since P19 ES cells are pluripotent and respond to external stimuli that have the potential to alter the phenotype and thus its stemness, other cell types used in RNA silencing studies do not display the obvious effect and therefore, may affect experiments in subtle ways that would go undetected. This study for the first time provides evidence that raises concern and warrants extreme caution while using the pLKO.1-puro control shRNA vector because of its unexpected non-specific effects on cellular integrity., (Published by Elsevier Inc.)

Luman (also known as LZIP or CREB3) is a transcription factor and a member of the cAMP responsive element-binding (CREB) family proteins. Although Luman has been detected in apoptotic granulosa cells and disorganized atretic bodies, the physiological function of Luman in follicular development has not been reported. Our objective is to determine the role of Luman in folliculogenesis by knocking down Luman expression in mouse GCs (granulosa cells) using shRNA. Luman expression was successfully knocked down in mouse GCs at the mRNA and protein level, as confirmed by real-time quantitative PCR, western blot and immunofluorescence staining, respectively. Knockdown of Luman significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in cell culture medium. Furthermore, Luman knockdown promoted cell proliferation but had no effect on cell apoptosis. To elucidate the regulatory mechanism underlying the effects of Luman knockdown on steroid synthesis and cell cycle, we measured the mRNA and protein expression levels of several related genes. The expression of Star, Cyp19a1, and Cyp1b1, which encode steroidogenic enzymes, was down-regulated, while that of Cyp11a1 and Runx2, which also encode steroidogenic enzymes, was up-regulated. The expression of the cell cycle factors Cyclin A1, Cyclin B1, Cyclin D2, and Cyclin E was significantly up-regulated. Among apoptosis-related genes, only Bcl-2 was down-regulated, while Caspase 3, Bax and p53 were not significantly affected, suggesting that Luman knockdown may regulate cell cycle activity and hormone secretion at the transcriptional and translational level in mouse GCs. The expression of two important genes associated with folliculogenesis in mouse GCs, Has2 and Ptgs2, were also significantly altered by Luman knockdown. In conclusion, the findings of this study indicate that Luman regulates mouse GCs modulation of steroid synthesis, cell cycle activity and other regulators of folliculogenesis., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: Osteoarthritis (OA) is a chronic, incurable and destructive joint disease that is characterized by chondrocyte hypertrophy and cartilage degradation. Angiogenesis, mediated by the action of vascular endothelial growth factor (VEGF), is known to be a contributing factor in the pathogenesis of OA. In this study, we use a lentivirus-based approach to investigate whether VEGF knockdown would be beneficial to chondrogenesis and could prevent or slow down OA progression. We first profiled cytokines in human OA cartilage using cytokine antibody arrays. This revealed that as many as 21 angiogenesis-related cytokines were significantly upregulated in severe OA cartilage compared to mild OA samples. Next, we infected chondrocytes with VEGF small hairpin RNA (shRNA) lentivirus (LV-VEGF shRNA) and treated these cells with tumour necrosis factor alpha (TNF-α) to induce hypertrophy. The results showed that inhibition of VEGF not only enhanced chondrogenic differentiation, but also protected chondrocytes from TNF-α-induced hypertrophy. We also found that knockdown of VEGF suppressed TNF-α-induced phosphorylation of ERK1/2 in chondrocytes. Furthermore, using a surgically induced OA rat model, we showed that VEGF inhibition delayed OA progression in animals given intra-articular injection of LV-VEGF shRNA. In conclusion, in this study, we have shown that VEGF knockdown can enhance chondrogenesis and prevent OA progression, thus providing evidence that inhibition of VEGF may be a potential therapeutic approach for OA patients., Key Messages: Numerous pro-angiogenic factors are upregulated in severe OA cartilage. Inhibition of VEGF by shRNA protects chondrocytes from TNF-α-induced hypertrophy. Knockdown of VEGF suppresses TNF-α-induced phosphorylation of ERK1/2 in chondrocytes. VEGF inhibition delays OA progression in rat model in vivo. Inhibition of VEGF may be a potential therapeutic approach for OA patients.

Purpose: To determine if a second dose of a lentivirus mediated small hairpin RNA that inhibits Vegf-A gene expression (LV-shRNA-Vegf-A) can improve lumen vessel area (LVA) of the outflow vein of an arteriovenous fistula (AVF) and decrease venous neointimal hyperplasia., Materials and Methods: Chronic kidney disease was created in C57BL/6 mice; 28 days later, an AVF was created by connecting the right carotid artery to the ipsilateral jugular vein. Immediately after AVF creation, 5 × 10(6) plaque-forming units of LV-shRNA-Vegf-A or control shRNA was administered to the adventitia of the outflow vein, and a second dose of the same treatment was administered 14 days later. Animals were sacrificed at 21 days, 28 days, and 42 days after AVF creation for reverse transcription polymerase chain reaction and histomorphometric analyses., Results: By day 21, there was a 125% increase in the average LVA (day 21, P = .11), with a decrease in cell proliferation (day 21, P = .0079; day 28, P = .28; day 42, P = .5), decrease in α-smooth muscle cell actin staining (day 21, P < .0001; day 28, P < .05; day 42, P = .59), and decrease in hypoxic stress (day 21, P < .001; day 28, P = .28; day 42, P = .46) in LV versus control shRNA vessels., Conclusions: A second dose of LV-shRNA-Vegf-A administration results in a moderate improvement in LVA at day 21., (Copyright © 2016 SIR. Published by Elsevier Inc. All rights reserved.)

Emerging evidences suggest that cancer stem cells (CSCs) are responsible for tumor growth, metastasis and treatment resistance. Nanog is one of the transcription factors that are essential for stem cellular physiology process. Previous studies reported that Nanog was detected in breast cancer and other solid tumors and indicated that it has oncogenic characteristics. However, expression feature of Nanog in breast cancer stem cells (BCSCs) enriched population and its biological function in BCSCs is poorly understood. In this study, CD44 + CD24- fraction sorting with Fluorescence Activated Cell Sorter and mammosphere culture were used for enriching BCSCs. We report here that Nanog was highly expressed in CSCs-enriched population from the breast cancer cells, as well as stemness-associated genes. In addition, we employed the lentivirus-mediated shRNA targeting Nanog to investigate function of Nanog in BCSCs. We found that targeted inhibition of Nanog could suppress proliferation and colony formation in breast cancer cells. Further studies showed that targeted inhibition of Nanog resulted in a decrease of BCSCs activities, including mammosphere formation, CD44 + CD24- proportion and expressions of stemness-associated genes. These data therefore suggest that Nanog possesses important function in BCSCs and targeted inhibition of Nanog may provide a novel means of targeting and eliminating BCSCs.

Aim: To explore neurite growth/regeneration and spinal cord injury repair after PTEN silencing via lentivirus-mediated RNAi., Materials & Methods: Cortical neurons were seeded on or adjacent to chondroitin sulfate proteoglycans. The length, number and crossing behavior of neurites were calculated. Lentivirus was locally injected into spinal cord contusion rats. The functional recovery and immunohistochemical staining were analyzed., Results: Neurites with PTEN silencing exhibited significant enhancements in elongation, initiation and crossing ability when they encountered chondroitin sulfate proteoglycans in vitro. In vivo PTEN silencing improved functional recovery significantly, and promoted axon and synapse formation, but not scar formation., Conclusions: PTEN silencing may be promising for spinal cord injury repair.

RNA interference (RNAi) has become a powerful and commonly used genetic tool for studying gene function. Vector-based short hairpin RNAs (shRNAs) trigger long-lasting RNAi effects compared to chemically synthesized siRNAs especially when they are imbedded in a lentiviral vector. In this chapter, a cost-effective method for shRNA design and expression is described. The strategy uses a carefully selected shRNA loop sequence and an antisense-loop-sense stem structure. Single and multiple shRNAs were expressed from the same vector, and thus the current design strategy has a great application potential in genomics.

Objective: To construct lentiviral vector that interferes with Nanog, investigate its expression in human gastric cancer cell SGC-7901-transplanted nude mice, and explore the effect of shRNA-Nanog transfection on the growth of the xenograft in mice., Methods: Lentivirus carrying shRNA-Nanog was prepared by incorporating previously validated siRNA into Nanog gene specific lentiviral vectors. The models of human gastric cancer transplantation were constructed in nude mice. The mouse models were randomly divided into lentivirus-shRNA-Nanog transfection group (experimental group), GFP infection group (empty vector group) and uninfected group (control group). The tumor volume and mass changes were measured. Small animal in vivo imaging was employed to examine the fluorescent intensity and tumor metastasis. The Nanog protein expression was determined by Western blot analysis. The apoptosis of transinfected tumor cells was analyzed by TUNEL method. Results The gene sequencing demonstrated that the recombinant lentivirus carrying shRNA-Nanog was successfully established. In vivo imaging showed that 27 days after transfection, the total fluorescent intensity in the experimental group was significantly lower than that in the empty vector group or the uninfected group. The xenograft volume and mass in the experimental group decreased significantly as compared with those in the empty vector group or the uninfected group. Western blotting indicated that the expression of Nanog protein in the experimental group was significantly lower than that in the empty vector or uninfected group. TUNEL results revealed that the apoptosis rate in the experimental group was significantly higher than that in the other two groups. No statistically significant difference was found between the empty vector group and the uninfected group., Conclusion: We successfully established Nanog gene-shRNA expression vector and capsulated it as lentivirus particles, which could inhibit the growth of xenograft in nude mice.

Background and Objective: Nuclear factor-κB is an important transcription factor and is closely associated with a variety of malignant tumors. The biological behavior of lung tumor cells can be reversed by inhibiting the expression of NF-κBp65 directly or indirectly. Nuclear factor-κBp65 gene shRNA recombinant plasmids were constructed and then infected with A549 cells. New stable cell lines were selected, and the ability of migration and adhesion was identified., Methods: Both scramble control sequence and interference sequence (shRNA) of human nuclear factor-κBp65 were designed and synthesized to build recombinant plasmids, with BamH I site at the 5' end and Xho I and EcoR I sites at the 3' end. A549 cells were infected, and stable transfection strains were selected by puromycin. Western blot and qRT-PCR methods were applied to assess the interference efficient of NF-κBp65 and the protein expression level of IκBα. Transwell and MTT assays were carried out to analyze the ability of migration and adhesion of A549 cells separately., Results: Recombinant plasmids were successfully built, and A549/NF-κB p65 scramble and A549/NF-κB p65 shRNA stable transfection strains were also successfully screened. Both mRNA and protein expression levels of NF-κBp65 showed that A549/NF-κBp65 shRNA cells decreased compared with A549/NF-κB p65 scramble cells and A549 cells, whereas the protein level of IκBα significantly increased. Both migration and adhesion abilities were also reduced., Conclusions: In this study, both mRNA and protein expression levels of NF-κBp65 were effectively suppressed by RNA interference technique. NF-κBp65 inhibition can significantly reduce the migration and adhesion ability of A549 cells.

Nerve growth factor (NGF) is involved in nerve sprouting, hyper-innervation, angiogenesis, anti-apoptosis, and preservation of cardiac function after myocardial infarction (MI). Positively modulating NGF expression may represent a novel pharmacological strategy to improve post-infarction prognosis. In this study, lentivirus encoding NGF short interfering RNA (siRNA) was prepared, and MI was modeled in the rat using left anterior descending coronary artery ligation. Rats were randomly grouped to receive intramyocardial injection of lentiviral solution containing NGF-siRNA (n = 19, MI-SiNGF group), lentiviral solution containing empty vector (n = 18, MI-GFP group) or 0.9% NaCl solution (n = 18, MI-control group), or to receive thoracotomy and pericardiotomy (n = 17, sham-operated group). At 1, 2, 4, and 8 wk after transduction, rats in the MI-control group had higher levels of NGF mRNA and protein than those in the sham-operated group, rats in the MI-GFP group showed similar levels as the MI-control group, and rats in the MI-SiNGF group had lower levels compared to the MI-GFP group, indicating that MI model was successfully established and NGF siRNA effectively inhibited the expression of NGF. At 8 wk, echocardiographic and hemodynamic studies revealed a more severe cardiac dysfunction in the MI-siRNA group compared to the MI-GFP group. Moreover, rats in the MI-siRNA group had lower mRNA and protein expression levels of tyrosine hydroxylase (TH) and growth-associated protein 43-positive nerve fibers (GAP-43) at both the infarcted border and within the non-infarcted left ventricles (LV). NGF silencing also reduced the vascular endothelial growth factor (VEGF) expression and decreased the arteriolar and capillary densities at the infarcted border compared to the MI-GFP group. Histological analysis indicated a large infarcted size in the MI-SiNGF group. These findings suggested that endogenous NGF silencing attenuated sympathetic nerve sprouting and angiogenesis, enlarged the infarct size, aggravated cardiac dysfunction, and potentially contributed to an unfavorable prognosis after MI.

Hemodialysis vascular access can develop venous neointimal hyperplasia (VNH) causing stenosis. Recent clinical and experimental data has demonstrated that there is increased expression of a disintegrin and metalloproteinase thrombospondin motifs-1 (ADAMTS-1) at site of VNH. The experiments outlined in the present paper were designed to test the hypothesis that targeting of the adventitia of the outflow vein of murine arteriovenous fistula (AVF) using a small hairpin RNA that inhibits ADAMTS-1 expression (LV-shRNA-ADAMTS-1) at the time of fistula creation will decrease VNH. At early time points, ADAMTS-1 expression was significantly decreased associated with a reduction in vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-9 (MMP-9) (LV-shRNA-ADAMTS-1 transduced vessels vs. controls). These changes in gene and protein expression resulted in favorable vascular remodeling with a significant increase in mean lumen vessel area, decrease in media/adventitia area, with a significant increase in TUNEL staining accompanied with a decrease in cellular proliferation accompanied with a reduction in CD68 staining. Collectively, these results demonstrate that ADAMTS-1 transduced vessels of the outflow vein of AVF have positive vascular remodeling.

The characterization of RNA interference and the accompanying microRNAs (miRs), together with the exogenous expression of artificial miR-like elements, has led to the development of strategies for specific and potent gene silencing. In turn, this allows manipulation of gene expression levels for target validation purposes in cell culture or for the generation of animal models. In this study we determined the optimal strategy to achieve the most potent knockdown using miR-based viral vectors. We studied polycistronic miRs in a viral vector context and evaluated knockdown potency of multiple-miRs targeting the same seed sequence in parallel with miRs targeting different seed sequences, both for a reporter and endogenous mRNA targets. We demonstrate that potent knockdown can be obtained in vitro and in vivo using viral vectors that encode a single miR-based short-hairpin RNA and report a generic and effective cloning platform for artificial miR30-based short-hairpin RNAs to generate potent knockdown viral vectors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Extracellular signal-regulated kinase (ERK)-2 is presumed to play an important role in the development of post-traumatic joint contractures. Using a rat injury model, we investigated whether treatment with ERK2 small interfering RNA (siRNA) could reduce the extent of joint capsule fibrosis after an induced injury. Rats were separated into three groups (n = 32 each): non-operated control group, operated contracture group and contracture-treatment group. Stable post-traumatic joint contracture was created through surgical intra-articular joint injury followed by eight weeks of immobilization. In the contracture-treatment group, the rats were treated with lentivirus (LV)-mediated ERK2 siRNA at days 3 and 7 post-surgery. The posterior joint capsule was assessed by western blotting, immunohistochemistry and biochemical analysis for changes in ERK2, phosphorylated (p)-ERK2, myofibroblast, total collagen and relative collagen Type III expression level. Biomechanical testing was used to assess the development of flexion contractures. Statistical analysis was performed using an analysis of variance. In the operated contracture group, rats that developed flexion contractures also showed elevated phosphorylated p-ERK2 expression. In the contracture-treatment group, ERK2 siRNA significantly reduced p-ERK2 expression levels, as well as the severity of flexion contracture development (p < 0.01). Myofibroblast numbers and measurements of total collagen content were also significantly reduced following ERK2 siRNA (p < 0.01). Relative collagen type III expression as a proportion of total of Types I and III collagen, however, was significantly increased in response to ERK2 siRNA (p < 0.01). Our findings demonstrate a role for ERK2 in the induction of joint capsule fibrosis after injury. Furthermore, we show that development of flexion contractures and the resultant increase of joint capsule fibrosis can be reduced by LV-mediated ERK2 siRNA treatment.

High-throughput short-hairpin RNA (shRNA) lentivirus screening is a powerful tool for identifying multiple functional regulators in embryonic stem cells (ESCs). shRNA libraries can efficiently down-regulate target genes persistently with high efficiency. The concurrent measurement of relative cell number by alamarBlue (AB) assay and undifferentiated ESC markers via an alkaline phosphatase (ALP) activity assay in the same cell culture well provides an efficient and economical way to pinpoint factors crucial for ESC pluripotency and/or expansion. Most of the renewal pathways affect ALP activity. Thus, multiple positive and negative regulators can be identified by this method. In addition, morphological changes and/or the expression levels of specific pluripotency or differentiation markers examined by immunofluorescence can be used as secondary screens for target-gene selection. In summary, we describe an efficient way to identify multiple regulators of ESC renewal using shRNAs. Curr. Protoc., (Copyright © 2013 John Wiley & Sons, Inc.)

SMC1A is a member of cohesin complex which has essential functions in cell cycle progression and DNA repair. Therefore, we choose SMC1A as a target gene therapy of glioblastoma. It is well known that glioblastoma has very low survival rate because of ineffectiveness of conventional treatments. This study was designed to explore the possibilities of small interfering RNA (siRNA)-mediated SMC1A silencing as alternative method of treatment. We found that the lentivirus-mediated RNAi system efficiently decreased the expression level of SMC1A. Inhibiting SMC1A expression efficiently (P < 0.001) resulted in inhibiting the proliferation and colony formation of U251 and U87MG cells. Moreover, we found that SMC1A silencing led to S cell-cycle arresting. Collectively, these results demonstrated the possibility of siRNA-mediated silencing of SMC1A as a therapeutic tool for the treatment of glioblastoma.

The present study aimed to find whether PROX1 is expressed in small cell lung cancer (SCLC) cell lines, and whether PROX1 knockdown with shRNA via lentivirus resulted in decreased cell proliferation. SCLC cell lines H69, H82, H187 and H889 were selected for the study. PROX1 mRNA and protein levels were determined with real-time reverse-transcription polymerase chain reaction(RT-PCR) and western blot, respectively. The localization and distribution of PROX1 was mapped by immunocytochemistry with a specific antibody. Three pairs of shRNA were selected from a pool of shRNA pairs, and packaged into lentivirus particles to infect the above cell lines. The non-target sequence (NT) and a house-keeping gene, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), were employed as controls. SCLC cell proliferation rates were measured with bromine deoxyuridine (BrdU) incorporation method. The results indicated levels of that PROX1 mRNA were detected in SCLC cell lines in the following rank order H69>H889>H187>H82. A similar profile for PROX1 protein expression was captured. The majority of PROX1 was concentrated at the cell nucleus. H69 was selected to represent the above SCLC cell lines. The PROX1 level in H69 cells was successfully reduced with shRNA lentivirus, and the cell proliferation rate of infected H69 cells was dramatically reduced by 20-50%. Hence, it is concluded that PROX1 expression in SCLC cell line is high, and can be reduced with shRNA lentivirus, thereby reducing the cell proliferation rate.

Small interfering RNA (siRNA)-induced RNA degradation can specifically inhibit viral infection and has been extensively investigated for its efficacy as an antiviral therapeutic approach. In this study we constructed a lentivirus vector carrying a U6-short hairpin RNA expression cassette to express siRNAs in vero cells. The lentivirus vector also expressed an enhanced green fluorescence protein as a reporter. Stable siRNA-expressing cell lines were successfully established, and the inhibition efficiencies of rationally designed siRNAs targeting conserved genomic regions of the Newcastle disease virus, an important disease of poultry world wide, were assessed. Our results showed that siRNAs targeting the nucleoprotein and matrix gene potently inhibited viral replication. Our study indicates that lentivirus-mediated delivery of siRNA and the resulting gene silencing can be used to study the functions of genes in viral replication and may have a potential transgenic antiviral application in poultry.

The etiology of osteoarthritis (OA) is complex and multifaceted. Osteoarthritis is a chronic and progressive disease of the joints that is characterized by the degradation of articular cartilage. A disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) is the major aggrecanase in cartilage. The aim of this study was to evaluate the effect of ADAMTS-5 knockdown on cartilage degradation. Rat articular chondrocytes were transfected with lentivirus‑mediated ADAMTS-5 small interfering RNA (siRNA) or with empty vector control plasmid DNA (as the control). The suppression efficiency was measured using real-time polymerase chain reaction (RT-PCR) and western blot analysis. We then selected the most effective siRNA (siRNA1) and constructed the lentivirus-mediated siRNA targeting ADAMTS-5 for stable transfection. An animal model of OA was created using male Sprague-Dawley rats. OA was induced by performing anterior cruciate ligament transection (ACL-T) and partial medial meniscectomy (PM). The animals (n=80, weight 250‑300 g) received an intra‑articular injection of the empty vector control plasmid DNA or lentivirus‑mediated ADAMTS-5 siRNA1 (20 µl, 1x108 TU/ml). The progression of OA was analyzed using Osteoarthritis Research Society International (OARSI) scores. Compared with the control, ADAMTS-5 gene expression was decreased by approximately 80% by siRNA1 in a monolayer culture of chondrocytes. The intra‑articular injection of lentivirus‑mediated ADAMTS-5 siRNA1 in vivo resulted in the downregulation of ADAMTS-5 protein expression and improved OARSI scores (p<0.05). A single injection of lentivirus‑mediated ADAMTS‑5 siRNA prevented the degradation of articular cartilage. This method may provide a novel therapeutic strategy for the treatment of human OA.

Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 promotor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively,with ratios of 89.20% and 62.00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the combinational lentiviral vector could inhibit virus replication efficiently.

Objective: RNA interference technology (siRNA) was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma SK-MES-1 cells, and the cell biological behaviors were investigated to explore the function of DJ-1 gene., Methods: A targeted DJ-1 siRNA lentiviral vector with a green fluorescent protein (GFP) as a reporter was constructed. The constructed DJ-1 siRNA and control-siRNA vectors were infected into SK-MES-1 cells as experimental (DJ-1 siRNA) and control (Control siRNA) groups, respectively. The DJ-1 protein expression was determined by Western blot. The cell proliferation capability was measured with methyl thiazolyl tetrazolium (MTT). The cell cycle was analyzed by flow cytometry. The capability of cell migration was determined by Transwell method., Results: Compared with control-siRNA and blank-control groups, the protein expression of DJ-1 gene was down-regulated, the capability of cell proliferation was obviously inhibited (P<0.01), the cell cycle was arrested with increased number of G1- and G2-phase cells and reduced number of S-phase cells, and the capability of cell migration was significantly decreased (P<0.01) in the DJ-1 siRNA-infected cells., Conclusion: DJ-1 gene might play a role in promoting cell proliferation and cell migration capability in vitro in lung cancer SK-MES-1 cells.

Intestinal inflammatory disease is a kind of non-specific disease with morbidity increasing yearly. It has been proved that the Toll like receptor 4 (TLR4) signaling pathways are closely related to intestinal inflammatory diseases. Myeloid differentiation protein 88 (Myd88) is a critical adaptor protein of TLR4 signaling and critical for the study of intestinal inflammatory disease, but stable Myd88 knockdown in vitro models of cell line are still very few. In the present study, an HIV-1-based lentivirus three-plamid packaging system was used for the construction of a lentivirual vector mediating RNA interference (RNAi) against Myd88 in intestinal fossae epithelial cell line-6 (IEC-6). Real-time PCR and Western blot were used to detect Myd88 expression. Annexin V staining and flowcytometry (FLM) were applied to detect and evaluate the early apoptosis. The results showed that lentiviral vectors containing the shRNA expression cassette specifically targeting Myd88 were constructed and efficiently stably knocked down Myd88 expression in IEC-6 cell line. Early apoptosis was significantly decreased after Myd88 knockdown. This study successfully constructed a lentivirus-based RNAi for Myd88 and detailed the key technique combined with characteristics of the early apoptosis after the Myd88 knockdown, provided a novel, stable and repeatable in vitro model for the pathogenesis, targeting therapeutic study for the intestinal inflammatory diseases.

Objective: To design and construct a lentiviral vector containing shRNA against rat glucose-regulated protein 78 gene (GRP78), and to establish rat pancreatic acinar cell line with stable knockdown of GRP78 expression., Methods: Constructed three plasmid expression vectors coding shRNA against GRP78, and converted them into lentiviral particles using three plasmid package systems. Then, AR42J cells were transduced with produced lentiviral particles. The green fluorescent protein (GFP) positive cells were selected by fluorescence-activated cell sorting (FACS), the GRP78 gene mRNA and protein expression were detected by real-time PCR and Western blot., Results: The DNA sequencing showed that the lentiviral vectors containing shRNA against GRP78 gene were constructed correctly. After the transduction, highly efficient transduction (> 85%) of lentivirus in AR42J cells was observed by fluorescent microscopy and FACS. Quantitative real-time PCR showed that GRP78 mRNA expression in AR42J cells was suppressed by LVshGRP78-1, LVshGRP78-2 and LVshGRP78-3 lentivirus about 69.4% +/- 1.42%, 74.7% +/- 1.69% and 86.6% +/- 1.73% as compared with that of the untreated cells (P < 0.05), while LV-Non Target had no significant effect on the GRP78 mRNA level (P > 0.05). Western blot showed that the suppressed effect of LVshGRP78 lentivirus on GRP78 protein was consistent with GRP78 mRNA., Conclusion: The results demonstrated that lentiviral vectors containing the shRNA against GRP78 gene were successfully constructed, which could stably knock down the GRP78 expression in AR42J cells. This study will provide a new cell model for further study of the role of GRP78 in the pathogenesis of acute pancreatitis.

Aims/hypothesis: The physiological significance of growth factor receptor-bound protein-10 (GRB10) in the pancreas is unclear. We hypothesised that GRB10 is involved in pancreatic apoptosis, as GRB10 binds with a family of cell-survival-related proteins implicated in apoptosis., Methods: Lentiviral vector small hairpin RNA (shRNA) targeting Grb10 was injected in vivo via an intraductal pancreatic route to target pancreatic tissues in adult mice, which were studied 2 weeks post-injection., Results: Using the TUNEL assay, we demonstrated for the first time that in vivo injection of lentivirus shRNA Grb10 directly into the adult mouse pancreas induced apoptosis in both exocrine and endocrine (alpha and beta) cells. This effect was more pronounced in alpha cells. Levels of the pro-apoptotic protein BCL2-interacting mediator of cell death (BIM) in islets was higher in lentivirus shRNA Grb10 than in lentivirus shRNA scramble mice. In the apoptotic pathway, BIM initiates apoptosis signalling, leading to activation of the caspase cascade. We propose that, when complexed with GRB10, BIM is inactive. On activation by stress signalling or, in the present study, following injection of lentivirus shRNA Grb10 into pancreas, BIM becomes unbound from GRB10 and activates the caspase cascade. Indeed, caspase-3 activity in islets was higher in the experimental than in the control group. Apoptosis induced by shRNA Grb10 resulted in a 34% decrease in fasting plasma glucagon. Mice injected with shRNA Grb10 had improved glucose tolerance despite reduced insulin secretion compared with shRNA scramble control mice., Conclusions/interpretation: GRB10 is critically involved in alpha cell survival and, as a result, plays an important role in regulating basal glucagon secretion and glucose tolerance in adult mice.

Objective: The objective of the study was to explore the inhibitive role of cyclin D1 gene silence in laryngeal squamous cell carcinoma by lentivirus-mediated RNA interference., Materials and Methods: Cd1-RNAi-Lentivirus and the control lentivirus (GFP-Lentivirus) were transfected into Hep-2 cells. Reverse transcriptase polymerase chain reaction was performed to explore the cyclin D1 expression level in Cd1-RNAi-Lentivirus-transfected Hep 2 cells. The apoptosis and viability of Cd1-RNAi-Lentivirus-treated Hep-2 cells were measured with flow cytometry and methyl thiazolyl tetrazoliym assay. In an animal experiment, 10 mice bearing Hep-2 cell tumor were intratumorally injected with Cd1-RNAi-Lentivirus; and the other 10 mice were injected with GFP-Lentivirus. Terminal deoxytransferase-mediated dUTP nick end labeling stains and transmission electron microscope were used to observe the apoptosis in the xenografts., Results: Cyclin D1 was knocked down after Cd1-RNAi-Lentivirus was transfected into Hep-2 cells. The proliferative ability of Hep-2 cells was significantly inhibited by Cd1-RNAi-Lentivirus, and a significant apoptosis of Hep-2 cells was also observed after Cd1-RNAi-Lentivirus transfection. The average weight and volume of tumors in the Cd1-RNAi-Lentivirus-treated group were significantly lower than those in the control group (P < .01). The significant apoptosis was detected with terminal deoxytransferase-mediated dUTP nick end labeling stain and transmission electron microscope., Conclusions: The present findings suggest that cyclin D1 gene silence by lentivirus-mediated RNA interference can inhibit growth and promote apoptosis of laryngeal squamous cell carcinoma., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Abnormal gene transcription plays an important role in oncogenesis. In cancer cells, the improper activation of specific genes is usually ascribed to aberrant transcription machinery including transcription factors, RNA polymerase II, and Mediator complex. This study reports on short hairpin RNA (shRNA)-mediated gene silencing of MED19, a subunit of Mediator complex, and its effect on the growth of pancreatic cancer cells. RNA interference was performed by lentivirus shRNA system to specifically knockdown MED19 expression in Aspc-1 and Panc-1 cells. The knockdown efficiency of MED19 was confirmed by quantitative RT-PCR and western blot. The effect of MED19 shRNA on Aspc-1 and Panc-1 cell proliferation was evaluated by methylthiazoletetrazolium assay, BrdU incorporation assay, colony formation assay, and flow cytometry assay. This study shows that downregulation of MED19 remarkably reduced cancer cell proliferation and colony formation capacity in two pancreatic cancer cell lines. In addition, downregulated MED19 induced G1-phase cell cycle arrest and apoptosis. This study provides a potent role of MED19 in promoting pancreatic cancer growth and a possible drug target for cancer therapy.

The expression of CHOP (C/EBP homologous protein), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress induced by cyclic stretch and leads to rat AF cells apoptosis. However, whether the suppression of CHOP can inhibit apoptosis and attenuates disc degeneration by cyclic stretch remains unclear. The aim of this study was to evaluate the suppressive effects of lentiviral CHOP shRNA on apoptosis induced by cyclic stretch in rat annulus fibrosus (AF) cells in vitro and disc degeneration of rat lumber spine in vivo. Lentiviral CHOP shRNA was constructed and introduced into AF cells. After stretched by the Flexcell Tension Plus system with 20% elongation for 36 h, silencing of the CHOP gene was identified by RT-PCR and Western blot. Inhibition of apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining. The effect of CHOP shRNA on disc degeneration was determined in vivo by using a rat model. At 7 weeks after intradiscal injection of the control or CHOP shRNA in the L4/L5 and L5/L6 discs, disc degeneration was assessed by X-ray examination, magnetic resonance imaging (MRI) assessment, and HE and TUNEL staining. A significant decrease in CHOP mRNA and protein expression was detected in AF cells with CHOP shRNA transfection after 36 h stretch. There was a significant decrease in apoptotic incidence in cells treated with CHOP shRNA, which was parallel to the expression of CHOP. Injection of CHOP shRNA in vivo resulted in the improvement in MRI and histologic score, and decrease in the apoptosis in the disc. No significant change in disc height was observed. In conclusion, a novel lentiviral vector expressing CHOP shRNA efficiently inhibits apoptosis in rat AF cells by silencing CHOP expression. In a rat model, intradiscal injection of CHOP shRNA induces the suppression of disc degeneration. The therapeutic effects of lentiviral CHOP shRNA should be further explored.

RNA interference (RNAi)-based gene silencing is widely used in laboratories for gene function studies and also holds a great promise for developing treatments for diseases. However, in vivo delivery of RNAi therapy remains a key issue. Lentiviral vectors have been employed for stable gene transfer and gene therapy and therefore are expected to deliver a stable and durable RNAi therapy. But this does not seem to be true in some disease models. Here, we showed that lentivirus delivered short-hairpin RNA (shRNA) against human papillomavirus (HPV) E6/E7 oncogenes were effective for only 2 weeks in a cervical cancer model. However, using this vector to carry two copies of the same shRNA or two shRNAs targeting at two different but closely related genes (HPV E6 and vascular endothelial growth factor) was more effective at silencing the gene targets and inhibiting cell or even tumor growth than their single shRNA counterparts. The cancer cells treated with dual shRNA were also more sensitive to chemotherapeutic drugs than single shRNA-treated cells. These results suggest that a multi-shRNA strategy may be a more attractive approach for developing an RNAi therapy for this cancer.

RNA interference (RNAi), an evolutionarily conserved process of gene silencing, is now a common tool in gene functional studies. However, potential "off-target effects" is one of major concerns in RNAi experiment associated with false positive results. Apart from continuing improvement in small interfering RNA (siRNA) designs, there is no method available to prevent the generation of "off-target effects" resulted from possible identity between siRNA and abundant cellular mRNA transcripts. In the present study, we have developed a lentiviral vector-mediated system that allows efficient siRNA silencing and concurrent rescue of targeted genes. While this approach does not eliminate potential "off-target effects," concurrent rescue of a target gene allows a definite judgment with regard to a phenotype change, either from expected siRNA silencing or "off-target effects." The system has been validated with murine CIN85 gene and may be generally applicable in molecular studies from broad fields., (Copyright © 2011 Wiley Periodicals, Inc.)

Elevated heat shock protein 27 (Hsp27) expression has been found in a number of tumors, including breast, prostate, gastric, uterine, ovarian, head and neck, and tumor arising from the nervous system and urinary system, and determined to be a predictor of poor clinical outcome. Although the mechanism of action of Hsp27 has been well documented, there are currently no available inhibitors of Hsp27 in clinical trials. RNA interference (RNAi) has the potential to offer more specificity and flexibility than traditional drugs to silence gene expression. Not surprisingly, RNAi has become a major focus for biotechnology and pharmaceutical companies, which are now in the early stages of developing RNAi therapeutics, mostly based on short interfering RNA (siRNAs), to target viral infection, cancer, hypercholesterolemia, cardiovascular disease, macular degeneration, and neurodegenerative diseases. However, the critical issues associated with RNAi as a therapeutic are delivery, specificity, and stability of the RNAi reagents. To date, the delivery is currently considered the biggest hurdle, as the introduction of siRNAs systemically into body fluids can result in their degradation, off-target effects, and immune detection. In this chapter, we discuss a method of combined lentiviral and RNAi-based technology for the delivery and permanent silencing of the hsp25 gene.

Objective: For studying the functions of periostin (POSTN) further, the recombinant plasmid and lentiviral vector for human periostin were constructed. The mRNA and protein expressions of POSTN were tested after the infection with the shRNA plasmid and lentivirus of POSTN in primary keloid fibroblasts (KFb)., Methods: We designed a specific sequence of small hair RNA targeting POSTN gene which containing both sense and antisense oligo DNA of the targeting sequence. It was cloned into the vectors to construct a plasmid and a lentiviral vector. The plasmid was converted into the competent DH5α E.coli, which confirmed by PCR and sequencing. Then the viral vector and the packed systemic vector were co-transfected 293T cells to get the lentivirus. KFb was infected with the plasmid and lentivirus. The expressions of POSTN in KFb were detected by RT-PCR and Western blotting. MTT was used to examine the proliferation of KFb and SFb., Results: The expression of POSTN mRNA in KFb infected with plasmid reduced 43% compared with negative control cells (1.98±0.03 vs 1.12±0.03, F=688.291, P<0.001). The mRNA and protein levels of POSTN in KFb infected with the lentivirus reduced 46% (0.44±0.09 vs 0.81±0.07, F=90.06, P<0.001) and 45% (0.29±0.04 vs 0.53±0.09, F=33.43, P<0.001), respectively, compared with negative control cells. Compared with negative control cells, the decreased proliferation rates were 19%, 14%, 18% in 48 h, 72 h, 96 h, respectively in KFb infected with the lentivirus., Conclusion: The expression of POSTN in primary keloid fibroblasts reduced after infecting plasmid or lentiviral shRNA expression vector targeting human POSTN, which will be a basis to the further function study of POSTN.

Manipulation of gene expression is an invaluable tool to study gene function in vitro and in vivo. The application of small inhibitory RNAs to knock down gene expression provides a relatively simple, elegant, but transient approach to study gene function in many cell types as well as in whole animals. Short hairpin structures (shRNAs) are a logical advance as they can be expressed continuously and are hence suitable for stable gene knockdown. Drug-inducible systems have now been developed; however, application of the technology has been hampered by persistent problems with low or transient expression, leakiness or poor inducibility of the short hairpin, and lack of reversibility. We have developed a robust, versatile, single lentiviral vector tool that delivers tightly regulated, fully reversible, doxycycline-responsive knockdown of target genes (FOXP3 and MYB), using single short hairpin RNAs. To demonstrate the capabilities of the vector we targeted FOXP3 because it plays a critical role in the development and function of regulatory T cells. We also targeted MYB because of its essential role in hematopoiesis and implication in breast cancer progression. The versatility of this vector is hence demonstrated by knockdown of distinct genes in two biologically separate systems.

Objective: To construct a lentiviral expression vector for short hairpin RNA (shRNA) of human survivin gene, and assess its gene silencing effect in human ectopic endometrial cells., Methods: Human survivin gene shRNA sequence was designed using a software available on-line. The synthesized shRNA sequence was cloned into the pGCL-GFP vector to construct LV-survivin shRNA, which was confirmed by PCR and DNA sequence analysis. The packaging 293T cells were cotransfected with LV-survivin shRNA, pHelper 1.0 and pHelper 2.0, and the titer of the lentivirus was determined. The recombinant lentivirus was injected into human ectopic endometrial cells and the survivin mRNA expression was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in comparison with that in the non-transfected and blank vector-transfected human ectopic endometrial cells., Results: PCR analysis and DNA sequencing confirmed correct insertion of the shRNA sequence into the lentiviral vector. The titer of virus after packaging was 8x10(8) U/ml. Survivin mRNA expression in human ectopic endometrial cells transfected by LV-survivin shRNA was significantly inhibited compared with those in the non-transfected and empty vector transfected human ectopic endometrial cells (P<0.01), and no significant difference was found between the latter two groups., Conclusion: The lentiviral shRNA vector of survivin gene constructed can effectively inhibit the expression of survivin gene in human ectopic endometrial cells in vitro. This vector provides a tool for investigating the role of survivin gene in the occurrence and progression of endometriosis and for searching new therapeutic targets.

Peroxisome proliferator-activated receptordelta (PPARdelta), as a downstream target of adenomatous polyposis coli (APC) signaling pathway, has been presumed to play some roles in colorectal carcinogenesis. However, the exact role of PPARdelta in colorectal cancer remains unclear. An HIV-1-based lentivirus packaging system was used for the construction of a lentiviral vector (lentivector) mediating RNA interference against PPARB. The direct sequencing demonstrated that the resulting lentivector containing the short-hairpin RNA expression cassette specifically targeting PPARdelta (sh-PPARdelta) was successfully constructed, and designated as pLVshPPARdelta. The control vector was designated as pLVControl. After the transduction, we observed highly efficient transduction (> 90%) of lentivirus in KM12C cells by fluorescent microscopy and fluorescence-activated cell sorting. Quantitative RT-PCR showed that pLVshPPARdelta lentivirus reduced PPARdelta mRNA expression by about 70.0% in KM12C cells as compared with that of the untreated cells (P < 0.05), while pLVControl had no significant effect on the PPARdelta mRNA level (P > 0.05). Western blot revealed an obvious reduction of PPARdelta protein expression in pLVshPPARdelta treated cells and showed no obvious difference between the control group and the untreated group. The results demonstrated that the lentivector mediating RNAi against PPARdelta was successfully constructed, which could stably knock down the PPARdelta expression in KM12C cells. This study finally provided a new cell model for the study of PPARdelta's function in colorectal cancer.

Objective: To observe the effect of the lentiviral vectors expressing small interfering RNA (siRNA) for survivin gene knockdown in inhibiting Hep-2 cell growth in vitro and its tumorigenicity in nude mice., Methods: The tumorigenicity of Hep-2 cells transfected with the siRNA mediated by the lentiviral vectors was tested in nude mice. The expression of survivin gene of the transfected cells at the mRNA and protein levels were detected by RT-PCR and Western blotting, respectively, and the cell cycle changes were analyzed by flow cytometry., Results: Transfection of the siRNA targeting survivin significantly decreased the expression of survivin mRNA and protein in Hep-2 cells in vitro by 60%-85% and 70%, respectively, resulting also in increased cell apoptosis as shown by flow cytometry (P<0.01). The transfection significantly lowered the tumorigenicity of the cells in nude mice., Conclusion: The lentiviral vectors expressing survivin siRNA can significantly inhibit survivin gene expression in Hep-2 cells and induce the cell apoptosis in vitro, and suppress the tumorigenicity of the cells in nude mice.

Objective: To investigate whether RelB-silenced bone marrow-derived dendritic cells (BMDC) pulsed with torpedo acetylcholine receptor (TAChR) immuno-dominant peptide Talpha146~162 can induce tolerance in T cells primed with TAChR., Methods: Recombinant lentivirus that produced RelB siRNA and control lentivirus were prepared and used to infect BMDCs. The infected BMDCs were stimulated with LPS,and the resulting cells were designated as DC-siRelB or DC-control, respectively. The mRNA and protein expression of RelB were examined by quantitative real-time PCR and Western blot. Cell surface markers of DC were evaluated by flow cytometry. IL-12 in the supernatant was detected by ELISA. Mice were randomly divided into 6 groups: A1, A2, A3,K1, K2, and K3. On day 0, group A1, A2, and A3 were primed with TAChR in CFA and group K1, K2 and K3 were primed with KLH+CFA. On day 7, group A2 and K2 were injected with Talpha146~162 pulsed DC-siRelB, group A3 and K3 were injected with Talpha146~162 pulsed DC-control, while A1 and K1 group received PBS at the same time. On day 14, lymphocyte proliferative response of the 4 groups were measured., Results: Recombinant lentivirus including RelBshRNA genes was successfully constructed. RelB siRNA knocked down RelB expression in BMDCs obviously. Compared with DC-control, DC-siRelB expressed a significantly lower level of CD80, CD86, and MHC class II on their surface, producing lower level of IL-12. Compared with group A1 and A3, lymphocyte proliferative response to TAChR of A2 group was suppressed significantly (P<0.05). No different lymphocyte proliferative responses to KLH and ConA were seen in group A1, A2 and A3 (P>0.05). No different lymphocyte proliferative responses were seen in group K1, K2 and K3 (P>0.05)., Conclusion: Lentiviral-mediated RelB-silenced BMDCs are maturation resistant and can induce antigen-specific tolerance in TAChR primed C57BL/6 mice,which provides a basis for further study of their therapeutic potential in myasthenia gravis.

Objective: To construct a small interfering RNA (siRNA) expression vector targeting hepatoma-derived growth factor (HDGF) and establish a lung adenocarcinoma cell line stably expressing siRNA-HDGF., Method: RT-PCR was used to examine HDGF expression in lung adenocarcinoma samples and the matched adjacent lung tissues, and also in lung adenocarcinoma SPC-A-1 cell line. A recombinant lentivirus shRNA-HDGF vector was constructed and transfected into SPC-A-1 cells via Lipofectamine 2000, and the cells with stable expression of HDGF-siRNA was screened by blasticidin selection. The interference effect of siRNA-HDGF was assessed by real-time PCR., Results: Compared to the adjacent lung tissues, lung adenocarcinoma and SPC-A-1 cells showed increased expression of HDGF. The recombinant lentivirus shRNA-HDGF vector was successfully constructed and verified by sequence analysis. siRNA-HDGF recombinants markedly inhibited the expression of HDGF in SPC-A-1 cells., Conclusion: HDGF expression increases in lung adenocarcinoma and SPC-A-1 cell lines. The recombinant siRNA-HDGF lentivirus vector can inhibit the expression of HDGF in SPC-A-1 cells.

Objective: To investigate the effect of osteopontin silencing on the invasion and apoptosis of U251 cells., Methods: The invasion, apoptosis and levels of uPA, MMP-2 and MMP-9 were determined by invasion assay, flow cytometry, Western blot and real-time fluorescence quantitative PCR respectively., Results: Osteopontin small interference RNA (siRNA) inhibited osteopontin expression and cell invasion, promoted apoptosis in U251 cells. In addition, the expression of Bcl-2, uPA, MMP-2 and MMP-9 was decreased, while Bax level was elevated., Conclusion: Osteopontin siRNA can inhibit U251 cells invasion via the down-regulation of uPA, MMP-2 and MMP-9 levels, and promote apoptosis through induction of Bax expression and inhibition of Bcl 2 level. It suggests that osteopontin plays an important role in human glioma progression.

Aim: To investigate RNA interference targeting signal transducer and activator of transcription-3 (STAT3) on invasion of human pancreatic cancer cells., Methods: We constructed three plasmids of RNA interference targeting the STAT3 gene. After LV (lentivirus)-STAT3siRNA (STAT3 small interfering RNA) the vector was transfected into the human pancreatic cell line, SW1990 and cell proliferation was measured by the MTT assay. Flow cytometry was used to assess cell cycle. Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) mRNA and protein expression were examined by quantitative PCR and western blotting, respectively. The invasion ability of SW1990 cells was determined by cell invasion assay., Results: We successfully constructed the LV-STAT3siRNA lentivirus vector and proved that it can suppress expression of STAT3 gene in SW1990 cells. RNA interference of STAT3 by the LV-STAT3siRNA construct significantly inhibited the growth of SW1990 cells, in addition to significantly decreasing both VEGF and MMP-2 mRNA and protein expression. Moreover, suppression of STAT3 by LV-STAT3siRNA decreased the invasion ability of SW1990 cells., Conclusion: The STAT3 signaling pathway may provide a novel therapeutic target for the treatment of pancreatic cancer since it inhibits the invasion ability of pancreatic cancer cells.

Objective: To construct the recombinant lentivirus vector containing short hairpin RNA (shRNA) inhibited DREAM expression and to investigate the gene therapy of neuropathic pain by inhibiting the expression of DREAM gene by RNA interference., Methods: An effective short hairpin RNA targeting to rat DREAM was cloned into the plasmids on the base of Lentivirous vectors, pKCSHR-Puro/GFP, and both of the pKCSHR-Puro/GFP-DREAM and Lentivector package plasmids mix were transferred into the 293T cells. The culture supernatant was harvested, and the virus titer was detected 48 hours after transferring. Thirty-six sheer breed pathogen free adult Sprague Dawley rats were randomly divided into 6 groups (6 in each group): normal control group (N); sham-operated group (S); CCI group (C0 group):CCI model without any intervention; Saline control group (C1 group); empty vector control group (C2 group); and LV-shRNADREAM lentiviral vector treatment group (C3 group). The rats in the last 3 groups respectively accepted injection of normal saline, blank vector, LV-shRNADREAM lentiviral vector in the subarachnoid on the 7th day after CCI, and the pain behavior was observed after 3, 7, 10, 14, 21 d after CCI. Green fluorescent protein (GFP) expression was detected by fluorescence microscope and the contents of DREAM mRNA and DREAM protein were detected by Realtime PCR and Western blot respectively in the rat lumbar spinal cord., Results: The short hairpin RNA sequences targeting at rat DREAM were cloned into the vectors, and an entry clone and an expression clone were constructed successfully confirmed by sequence analysis. Lentiviral packaging was successful in 293 T cell line and the transfection titer of the lentivirus was 1 x 10(8) IFU/mL. LV-shRNADREAM lentivirus vector was transfected successfully in the rat spine with Intrathecal injection of LV-shRNADREAM. Compared with the other 3 groups, heat pain threshold and mechanical pain threshold in Group C3 improved significantly (P<0.01), and the expression of DREAM mRNA and DREAM protein in the lumbar spinal cord in Group C3 were lowered significantly (P<0.01)., Conclusion: Lentivirus vectors containing rat DREAM gene are constructed successfully, and lentivirus mediated shRNA can inhibit the DREAM expression in the rat spine, which may prove to be an effective method for neuropathic pain.

Objective: To construct a miR-155-based lentivirus vector to induce cyclooxygenase-2 gene silencing in nasopharyngeal carcinoma (NPC) cells by expressing anti-COX-2 shRNAmir., Methods: miR-155-based anti-COX-2 shRNAmir template was synthesized and inserted into pLVTHM plasmid. The recombinant pLVTHM/shRNAmir was transfected into 293FT cells for packaging the lentivirus vector. After infection with the lentivirus vector, the GFP-positive cells were screened by flow cytometry, and COX-2 mRNA level was detected by RT-PCR., Results: Restriction digestion and DNA sequencing confirmed successful construction of the anti-COX-2 vector pLVTHM/shRNAmir. A subline of C666-1 cells was established after infection with the lentivirus vector, and the COX-2 expression in the cells was stably silenced., Conclusion: The shRNAmir lentivirus vector constructed may serve as an effective COX-2 inhibitor, which may facilitate future studies of gene therapy of NPC.

Objective: To establish a nasopharyngeal carcinoma (NPC) cell line with stable EIF4G1 gene silencing induced by small interfering RNA (siRNA)., Methods: The EIF4G1 mRNA levels in 8 NPC cell lines including 5-8F, 6-10B, C666-1, CNE1, CNE2, HNE1, HONE1, and SUNE1 were detected by fluorescence quantitative RT-PCR (QRT-PCR). The recombinant lentivirus shRNA expression plasmid targeting EIF4G1 gene was packaged into mature lentivirus by 293FT cells and used to infect 5-8F cells. After blasticidin selection of NPC cells with constant expression of the EIF4G1-siRNA, the efficiency of EIF4G1 mRNA expression interference was determined using QRT-PCR., Results: The 8 NPC cell lines showed differential expression of EIF4G1 mRNA, among which 5-8F cells had the highest EIF4G1 expression. The recombinant lentivirus plasmid pLenti6/BLOCK-iT-DEST/EIF4G1-shRNA was successfully constructed and verified by PCR and sequencing. The EIF4G1 mRNA level of 5-8F cells infected with shRNA-EIF4G1 lentivirus was significantly reduced as compared with the negative control and the blank control cells., Conclusion: The recombinant lentivirus vector pLenti6/BLOCK- iT-DEST/EIF4G1-shRNA we constructed results in marked downregulation of EIF4G1 mRNA expression and constant expression of EIF4G1-siRNA after infection of 5-8F cells.

To construct the lentiviral RNA interference (RNAi) vector of rat CXCR4 gene, three target sequences were selected according to rat CXCR4 mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized. After phosphorylation and annealing, these double-strand DNA were cloned to Bgl II and Hind III sites of pSUPER. Then the product pRiCXCR4 was confirmed by electrophoresis and sequencing. Next, CXCR4 shRNA was cloned to a transfer vector of lentivirus, pNL-EGFP, and pNL-RiCXCR4-EGFP was produced. It was confirmed by digestion and sequencing that CXCR4 shRNA expression structure was correctly cloned to pSUPER and pNL-EGFP respectively. Three plasmids, pNL-RiCXCR4-EGFP, pHELPER and pVSVG were cotransfected into 293T to package lentivirus particles. The functional titer of obtained virus was determined by flow cytometry after transduction in 293T, the resulting functional titer of unconcentrated virus and concentrated virus were 6.4 x 10(4) TU/mL and 6.9 x 10(6) TU/mL respectively. After the rat mesenchymal stem cells (rMSCs) were transduced with the constructed lentiviral vectors, real-time RT-PCR, Western blotting and flow cytometry were used to evaluate the level of CXCR4 expression. Compared with control group, the CXCR4 mRNA expression were obviously suppressed in all three experimental groups (rMSCs-CXCR4a, rMSCs-CXCR4b, rMSCs-CXCR4c), especially the expression rate in rMSCs-CXCR4b group was reduced by 95.6%. The RNAi lentivirus vector of rat CXCR4 gene has been constructed successfully. This greatly facilitates the further studies such as evaluation the role of CXCR4 in rMSCs recruitment to damaged tissue.

Objective: To construct a recombinant lentivirus harboring RNA interference sequence targeting mouse CD99 antigen-like 2 (mCD99L2) gene and observe its infection efficiency of 293FT cells., Methods: Four pairs of small interfering RNAs (siRNAs) targeting mCD99L2 cDNA were designed, synthesized and linked to the lentivirus vector SD1259 to construct the lentivirus shuttle plasmids. After sequencing, the 4 lentivirus shuttle plasmids were transfected into 293FT cells in the presence of packaging plasmids. Forty-eight hours later, the supernatant was collected and the titer and infection efficiency of the recombinant lentivirus were determined according to the expression of the reporter gene enhanced green fluorescent protein (EGFP) under fluorescent microscope., Results: DNA sequencing demonstrated that mCD99L2 siRNAs were successfully cloned to the lentiviral vector SD1259. The titer of concentrated virus was 1x10(7)/ml in the supernatant of the infected cells., Conclusion: The recombinant lentivirus containing siRNA targeting mCD99L2 gene has been successfully constructed, which provide the basis for future establishment of visualized cell model and animal model of Hodgkin's lymphoma.

In order to explore the role of TNF-alpha in Niemann-Pick type C (NPC) disease, lentiviral-delivered RNA interference (RNAi) was used to silence the expression of murine TNF-alpha gene in vitro and in npc mice. Interference efficiency of the lentivirus expressing TNF-alpha-siRNA, previously constructed with the concentration of 2 x 10(8) ifu/mL, was determined by RT-PCR and ELISA in BV-2 cells and astrocytes. At the same time, the constructed Lenti-TNF-alpha-siRNA was intracerebroventricularly infused into 4-week old npc mice for a 4-week period, and the mice were divided into 3 groups: Lenti-TNF-alpha-siRNA (n=6), control lentivirus (n=6), and NPC mice without any intervention (n=4). By using immunohistochemistry and real-time PCR, the down-regulation of the target genes was detected. The Lenti-TNF-alpha-siRNA downregulated the expression of murine TNF-alpha gene efficiently in vitro and the interference efficiency was 66.7%. Lentivirus could be expressed stably for long-term in the npc mice brain. Immunohistochemistry and real-time PCR revealed that, as compared with non-intervention group and Lenti-control group, Lenti-TNF-alpha-siRNA efficiently down-regulated the expression of murine TNF-alpha gene with the interference efficiency being 66.9%. TNF-alpha-siRNA down-regulated the expression of TNF-alpha gene in vitro and in vivo, which provided a potential tool for studying and treating neurodegenerative diseases and TNF-alpha-related diseases.

This study was aimed to construct a lentiviral vector of RNA interfered (RNAi)-kir2ds4 gene. In accordance with study-confirmed effective sequence of siRNA targeting kir2ds4 gene, the complementary DNA containing both sense and antisense oligonuctide of the targeting sequence was designed, synthesized and inserted into pSicoR-GFP vector containing U6 promoter and GFP sequence. The resulting lentiviral vector containing kir2ds4 shRNA was named as LV-sh kir2ds4, and confirmed by PCR and sequencing. 293T cells were co-transfected with lentiviral vector LV-sh kir2ds4 and packaging system. All virus stocks were produced by Lipofectamine 2000-mediated transfection. The titer of virus was tested according to the expression level of GFP. As a result, PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of kir2ds4 was constructed successfully. The titer of virus tested by expression level of GFP was 6 x 10(8) TU/ml. It is concluded that the lentivirus RNAi vector of kir2ds4 has been successfully constructed.

We describe the protocols of using siRNAs, or shRNAs delivered by a lentiviral vector, as a means to silence cancer-causing genes. We use cervical cancer as a model to demonstrate the inhibition of the human papillomavirus (HPV) oncogenes E6 and E7 in cervical cancer cells by RNAi and inhibition of the cell growth in vitro and tumor growth in mouse models. The protocols include methods on siRNA and shRNA design, production of lentiviral-vectored shRNA, transfection or transduction of cervical cancer cells with siRNA or shRNA, and detection of the inhibitory effects of siRNA or shRNA both in vitro and in vitro.

Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells.