Construction and identification of an RNA interference lentivirus vector targeting the Ras homology C gene of melanoma cells

Background Melanoma has the highest mortality among all superficial malignant tumors.The poor prognosis is due to its high metastasis rate and the lack of therapeutic targets.As a molecular switch that controls tumor metastasis,Ras homology C （RhoC） has been correlated with tumor progression,especially tumor invasion and metastasis.However,little research has been done about the effects of RNA interference （RNAi） targeting RhoC on the invasion and metastasis of melanoma.In this study,we constructed an RNAi lentivirus vector targeting the RhoC gene of melanoma cells and identified its silencing effects on the RhoC gene.Methods Based on the RhoC gene encoding information,three pGPU6/GFP/Neo-short hairpin （shRNA） plasmids were constructed.After detecting their silencing effects on the RhoC gene of A375 cells,the most effective pGPU6/GFP/ Neo-shRNA plasmid was packed with lentivirus to construct the recombinant pLenti6.3-EGFP-453 targeting RhoC.The lentivirus vector was used to infect A375 cells,and then the expression of RhoC mRNA and protein were determined with real-time PCR and Western blotting.Results The plasmids pGPU6/GFP/Neo-shRNA 336,pGPU6/GFP/Neo-shRNA 453,and pGPU6/GFP/Neo-shRNA 680 were constructed.After they were transfected into A375 cells,the expressions of RhoC mRNA and protein were 1.47±0.26,1.13±0.16,1.39±0.11 and 70.98±9.21,50.67±6.06,65.77±4.06,respectively.pGPU6/GFP/Neo-shRNA 453 was the most effective sequence,and was used to successfully construct the pLenti6.3-EGFP-453 lentiviral vector targeting RhoC.pLenti6.3-EGFP-453 was used to infect A375 cells.The expression of RhoC mRNA and protein were 1.05±0.05 and 62.04±15.86 in the lentivirus group,4.21±0.24 and 220.86±24.07 in the negative lentivirus control group,and 4.63±0.32 and 257.39±12.30 in the normal control group respectively with the difference between the lentivirus group and the control groups being statistically significant （P ＜0.05）.Conclusion The successfully constructed pLenti6.3-EGFP-453 vector targeting the RhoC can effectively infect human melanoma A375 cells in vitro,and significantly inhibit the RhoC mRNA and protein expression.

Lentivirus-mediated shRNA interference targeting STAT3 inhibits human pancreatic cancer cell invasion

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 favor （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 LVSTAT3siRNA 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.

Construction and identification of recombinant lentivirus-mediated gene transfer system for rat transducer of regulated CREB activity 1

Objective： To construct a recombinant lentivirus vector which carries SD rat transducer of regulated CREB activity-1（TORC1） gene and examine its ability to express the TORC 1 gene in vitro. Methods： The coding sequence of SD rat TORC 1 gene was amplified using PCR and cloned into pGC-FU vector. 293T cells were transfected using Lipofectamine 2000 and packaged for the recombinant lentivirus particles. When the cloned sequence was identified to be right, the recombinant lentivirus particles were amplified in a large quantity. The titer of virus was determined by real-time PCR and the level of TORC1 expression was examined by Western blot. Results： The recombinant lentivirus vector carrying TORC1 was constructed successfully and could express TORC1 at a high level in 293T cells in vitro, and the titer determined by real-time PCR was 2 × 10^8 TU/ml. Conclusion： The recombinant lentivirus vector could express TORCl gene at a high level, and was very helpful in the study of exploring the effect of TORC1 on spinal cord injury.

NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line

AIM： To construct the recombinant lentivirus expression plasmid, pLenti6/V5-NT4 p53（N 15）-antennapedia （Ant）, and study its effect on HepG2 cells. METHODS： Plasmid pLenti6/V5-NT4 p53（N15）-Ant was constructed incorporating the following functional regions, including signal peptide sequence and proregion of neurotrophin 4, N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein, Ant. Hepatocellular carcinoma （HepG2） cells were used for transfection. 3-[4,5-climethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide （MI-I） assay, lactate dehydrogenase （LDH） release assay, transmission electron microscopy （TEM） and flow cytometric analysis （FCM） were employed to investigate the effects of LV-NT4（Si）- p53（N15）-Ant in vitro on HepG2 cells. In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4（Si）-p53（N15）-Ant on tumor growth in nude mice.RESULTS： LV-NT4（Si）-p53（N15）-Ant significantly suppressed the growth of HepG2 cells. MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4（Si）-p53（N15）-Ant than by LV-EGFP. The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%, respectively, 48 h after infection with LV-NT4（Si）-p53（N15）-Ant, and was 33.9% and 95.8%, respectively, 72 h after infection with LV-NT4（Si）-p53（N15）-Ant （P 〈 0.01）. Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, but no significant changes in HepG2 cells infected with LV-EGFR Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, with degraded membranes, resulting in necrosis. LDH release from HepG2 cells was analyzed at 24, 48, 72 and 96 h after infection with LV-NT4（Si）-p53（N15）-Ant and LV-EGFP, which showed that LDH release was significantly higher in LV-NT4（Si）-p53（N15）-Ant treatment group （682 IU/L） than in control group （45 IU/L, P 〈 0.01）. The longer the time was after infection, the bigger the difference was in LDH release. FCM analysis showed that LV-NT4（Si）- p53（N15）-Ant could induce two different kinds of cell death： necrosis and apoptosis, with apoptosis being the minor type and necrosis being the main type, suggesting that LV-NT4（Si）-p53（N15）-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis. The in vivo study showed that LV-NT4（Si）-p53（N15）-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight. CONCLUSION： LV-NT4（Si）-p53（N15）-Ant is a novel recombinant lentivirus expression plasmid and can be used in gene therapy for cancer.

An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo

Background： Male germline stem cells（MGSCs） are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals.Method： The present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein（e GFP） transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice（1.5 to 2.0-month-old）.Results： Sixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eG FP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of g DNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter,suggesting e GFP transgene was suppressed by DNA methylation in vivo.Conclusion： This research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.

Construction and functional activity of a recombinant vector expressing rat glutamic acid decarboxylase 65

Objective Glutamic acid decarboxylase 2（GAD65） is a gamma-aminobutyric acid（GABA） synthetase.This study aimed to construct a recombinant lentivirus-rGAD65（rLV-rGAD65） vector containing the cDNA of rat GAD65（rGAD65） and assess its functional activity in vitro and in vivo.Methods cDNA of rGAD65 was amplified by RT-PCR and subcloned into the LV vector,forming the rLV-GFP-rGAD65 plasmid.The recombinant lentivirus particles（rLVrGAD65） were packaged by the LV Helper-Free System and the titer was measured.Primary rat lung fibroblasts were transfected with rLV-rGAD65.The expression of rGAD65 in fibroblasts was detected by immunocytochemistry and western blot and the level of GABA in the medium was assessed by high-performance liquid chromatograph（HPLC）.In vivo,rLV-rGAD65 was injected into the subthalamic nucleus（STN） of Sprague-Dawley rats using stereotaxic methods,and rGAD65 protein levels in the STN were assessed by immunohistochemistry and Western blot,while the GABA concentration in the substantia nigra pars reticulata（SNr） was assayed by HPLC.Results The sequence of rGAD65 cDNA was in accord with that in GenBank.The amino-acid sequence of rGAD65 had no mutations and the titer of rLVrGAD65 reached 6.8 × 108/mL.The efficiency of infection of fibroblasts was 80%,and the concentration of GABA in the medium was（48.14 ± 9.35） nmol/L.In vivo,rGAD65 expression was detected in the STN,and the concentration of GABA in the SNr increased from（5.95 ± 1.09） to（12.44 ± 3.79） nmol/g tissue.Conclusion The recombinant LVGFP-rGAD65 vector was successfully constructed.rLV-rGAD65-infected primary fibroblasts in vitro and the expressed rGAD65 catalyzed the formation of GABA from glutamic acid.In vivo,the concentration of GABA in the SNr was increased after rLV-rGAD65 injection into the STN.

In vivo Inhibitory Effect of Lentivirus-mediated RNA Interference Targeting RhoC on Growth of SKOV3 Cells

To investigate the inhibitory effect of lentivirus-mendiated RNA interference targeting RhoC on the growth of SKOV3 cells（ovarian cancer SKOV3 cells） in vivo, the vector expressing RNA interference targeting RhoC gene（LV-shRhoC） was constructed and the virus particles were packaged. The infection effiency of SKOV3 cells by the virus was estimated by green fluorescent protein expression on a fluorescence microscope and the expression of RhoC gene in the SKOV3 cells was detected by reverse transcription real time polymerase chain reaction（PCR）. Furthermore, human ovarian cancer SKOV3 cells, empty vector infected SKOV3 cells and interfered-vector infected SKOV3 cells were respectively seeded into nude mice, and the shape, mass, volume and histophathological changes of the transplanted tumors were observed 20 d later the mice were sacrified. The results show that lentivirus packa-ging particles can effectively infect SKOV3 cells and the lentivirus-mediated RNA interference can significantly in- hibit the expression of RhoC gene in SKOV3 ceils, the mass and volume of the transplanted tumor in the mice of the specific-control group（Lv-shRhoC） are all lower than the corresponding ones in the mice of negative- and blank-control groups（Lv-NC and SKOV3）. Moreover, the histopathlosical secion investigation shows that the nuclear Karyotype and histopathologic mitotic figure of SKOV3 cells in mice of the specific-control group are clearly lower than those in the mice of the negative-control group.Thus it is concluded that silencing RhoC gene by means of lenti- virus-mediated RNA interference targeting RhoC can obviously inhibit the growth of ovarian cancer cells（SKOV3） in vivo, which is a new strategy for the gene therapy of ovarian cancers.

Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene

Leptin resistance is a main mechanism of acquired childhood obesity,and the suppression of long form of leptin receptor(OBRb)gene expression in diet-induced obese rats indicates that the down-regulation of OBRb gene expression plays a pivotal role in the mechanism of leptin resistance.The aim of the present study was to construct the lentiviral RNA interference(RNAi)vector of rat OBRb gene and evaluate the effects of siRNA on silencing OBRb gene expression.The target sequence of siRNA-OBRb was designed,and the com-plementary DNA containing both sense and antisense oligonucleotides was synthesized.After phosphorylation and annealing,these double-stranded DNA was cloned to pRNA-lentivector-VGFP to construct pRNA-Lenti-OBRb-VGFP recombinants with U6-containing promoter,target sequence and Poly III terminator.Then,the products were confirmed by electrophoresis and sequencing analy-sis,and the effects of RNAi on reducing gene expression were further confirmed by real-time polymerase chain reaction in transfected rat glioma cells expressing OBRb.The target sequence of siRNA-OBRb was successfully cloned to pRNA-lentivector-VGFP,and the RNAi protocol specifically reduced the expression of OBRb mRNA by approximately 80%compared with controls in transfected rat glioma cells.The successful construction of rat lentivirus vectors expressing OBRb-specific shRNA may be useful for further investigation in vivo.

Inducible regulation of GDNF expression in human neural stem cells

Glial cell derived neurotrophic factor （GDNF） holds promises for treating neurodegenerative diseases such as Parkinson＇s dis- ease. Human neural stem cells （hNSCs） have proved to be a suitable cell delivery vehicle for the safe and efficient introduction of GDNF into the brain. In this study, we used hNSCs-infected with a lentivirus encoding GDNF and the hygromycin re- sistance gene as such vehicles. A modified tetracycline operator 7 （tetO7） was inserted into a region upstream of the EFI-α promoter to drive GDNF expression. After hygromycin selection, hNSCs were infected with a lentivirus encoding a KRAB-tetracycline repressor fusion protein （TTS）. TTS bound to tetO7 and suppressed the expression of GDNF in hNSCs. Upon administration of doxycycline （Dox） the TTS-tetO7 complex separated and the expression of GDNF resumed. The hNSCs infected with GDNF expressed the neural stem cell specific markers, nestin and sox2, and exhibited no significant change in proliferation rate. However, the rate of apoptosis in hNSCs expressing GDNF was lower compared with normal NSCs in response to actinomycin treatment. Furthermore, a higher percentage of Tuj-I positive cells were obtained from GDNF-producing NSCs under conditions that induced differentiation compared to control NSCs. The inducible expression of GDNF in hNSCs may provide a system for the controllable delivery of GDNF in patients with neurodegenerative diseases.

CCDC67基因慢病毒表达载体的构建及鉴定

目的 构建CCDC67基因慢病毒表达载体，为后续转染目的细胞奠定基础，并实现在细胞中高效、稳定表达。方法 从含有CCDC67基因的cDNA文库中，利用PCR方法钓取CCDC67基因编码区片段。将目的基因与酶切载体pGV-208进行定向连接，其产物转化细菌感受态细胞。对长出的克隆先进行菌落PCR鉴定，再对PCR鉴定阳性的克隆进行测序和比对分析，对比正确的克隆即为构建成功的目的质粒。将构建成功的目的质粒和两种辅助包装质粒共转染293T细胞，包装成慢病毒，在荧光显微镜下观察慢病毒转染293T细胞后荧光表达情况。采用Western blot检测CCDC67及绿色荧光蛋白融合蛋白表达情况。结果 PCR产物经电泳分析表明成功获取CCDC67基因cDNA克隆，测序结果显示慢病毒转染质粒连接构建正确。荧光表达检测显示293T细胞中产生慢病毒颗粒，Western blot显示CCDC67在细胞内稳定表达。结论 成功构建CCDC67基因慢病毒表达载体，为后续转染目的细胞后从分子水平探讨CCDC67基因功能奠定了实验基础。

Roles of FOXP3 in experimental atherosclerosis of ApoE-knockout mice

Background Subtypes of T cells, called regulatory T cells （Treg cell）, play a critical role in limiting autoimmune processes and inflammatory responses, The aim of this study was to explore functional roles of FOXP3 in the manifestation of atherosclerosis in Apolipoprotein E deficient （ApoE）-/- mice. Methods Lentivirus-mediated （siRNA） was used to knock down FOXP3 and FOXP3high＋CD4＋ CD25＋ T cells adoptive transfer assays in high fat diet ApoE-/- mice were done. The resulting atherosclerotie lesions were assessed by determining FOXP3 transcript levels and investigating the expression of FOXP3 protein in different tissues. Results Animals treated with siRNA of FOXP3 showed a significant increase in atherosclerotic lesion formation and a reduction in the number of FOXP3＋CD4＋CD25＋ T cells compared with other groups. Transfer of FOXP3highCD4＋CD25＋ T cells significantly decreased atherosclerotic plaque formation and increased the number of FOXP3＋ CD4＋ CD25＋ T cells. FOXP3 protein levels and FOXP3 transcript levels were lowest in the siRNA group, and were highest in tissues from the Treg transfer group. Conclusion FOXP3 plays an important role in regulating the inflammatory response within the atherosclerotic lesion. It can inhibit significantly the progression of the atherosclerosis plaque in ApoE-/- mice.