Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by...Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.展开更多
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 tar...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.展开更多
Lentivirus carrying the Atohl gene can infect Corti＇s organ and express a hair-like cell surface marker in the supporting cell area. However, expression of the gene carried by adenovirus is instantaneous, which undou...Lentivirus carrying the Atohl gene can infect Corti＇s organ and express a hair-like cell surface marker in the supporting cell area. However, expression of the gene carried by adenovirus is instantaneous, which undoubtedly limits its clinical application. Lentivirus acts as a carrier that can stably and continuously express genes. In this study, the cochlear structure and hearing level were not affected, and Atohl gene carried by lentivirus promoted the production of hair-like cells in the cochlear supporting cell area. This led to expression of the hair-like cell surface marker myosin 7a 30 days after lentivirus carrying Atohl was microinjected into the cochlear round window of rats.展开更多
AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mecha...AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated REDO gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.展开更多
Objective To explore the feasibility for therapy of spinal cord injury （SCI） by genetic engineering neural stem cell （NSC） modified by lentiviral vector. Methods Following the construction of the genetic engineer...Objective To explore the feasibility for therapy of spinal cord injury （SCI） by genetic engineering neural stem cell （NSC） modified by lentiviral vector. Methods Following the construction of the genetic engineering NSC modified by lentivirus to secrete both neurotrophic factor-3 （NT-3） and green fluorescence protein （GFP）, hemisection of spinal cord at the level of T10 was performed in 56 adult Wistar rats that were randomly divided into 4 groups （ n = 14 ）, namely 3 therapeutic groups and 1 control group. The therapeutic groups were dealed with NSC, genetic engineering NSC, and concentrated lentiviral supematant which carries both GFP and NT-3, respectively. Then used fluorescence microscope to detect the transgenic expression in vitro and in vivo, migration of the grafted cells in vivo, and used the Basso, Beattie, and Bresnahan （BBB） open-field locomotor test to assess the recovery of function. Results The transplanted cells could survive for long time in vivo and migrate for long distance. The stable transgenie expression could be detected in vivo. The hindlimb function of the injured rats in 3 therapeutic groups, especially those dealed with genetic engineering NSC, improved obviously. Concision It is feasible to combine NSC with lentivirus for the repair of SCI. NSC modified by lentivirus to deliver NT-3, acting as a source of neurotrophic factors and function cell in vivo, has the potential to participate in spinal cord repair.展开更多
In the present study, we constructed a lentivirus, FIV-CMV-GFP-miR-7-3, containing the microRNA-7-3 gene and the green fluorescent protein gene, and used it to transfect human glioma U251 cells. Fluorescence microscop...In the present study, we constructed a lentivirus, FIV-CMV-GFP-miR-7-3, containing the microRNA-7-3 gene and the green fluorescent protein gene, and used it to transfect human glioma U251 cells. Fluorescence microscopy showed that 80% of U251 cells expressed green fluorescence. Real-time reverse transcription PCR showed that microRNA-7-3 RNA expression in U251 cells was significantly increased. Proliferation was slowed in transfected U251 cells, and most cells were in the G1 phase of the cell cycle. In addition, the expression of the serine/threonine protein kinase 2 was decreased. Results suggested that transfection with a lentivirus carrying microRNA-7-3 can effectively suppress epidermal growth factor receptor pathway activity in U251 cells, arrest cell cycle transition from GI phase to S phase and inhibit glioma cell growth.展开更多
Cyclin-dependent kinase 2-associated protein 1（CDK2AP1）, a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, no studies on th...Cyclin-dependent kinase 2-associated protein 1（CDK2AP1）, a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, no studies on the function of CDK2AP1 in human lung cancer have been yet reported. In this study, overexpressing lentiviral vectors containing full-length CDK2AP1 cDNA and CDK2AP1 shRNA（short hairpin RNA） were constructed. Our results show that infecting A549 cells with lentivirus containing CDK2AP1 shRNA or full-length CDK2AP1 cDNA results in significantly down- or up-regulated the expression of CDK2AP1, respectively, thereby providing reliable tools for studying the function of CDK2AP1 in pulmonary carcinogenesis. Our data of MTT assay and flowcytometric analysis demonstrate that CDK2AP1 plays an important role in the proliferation/growth and cell cycling of A549 cells in vitro, and further investigation into its underlying mechanism of pulmonary carcinogenesis is needed.展开更多
AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We construct...AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-xL and bcl-xS were detected with western blot. RESULTS: We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved indown-regulating bcl-xL and up-regulating bcl-xS. CONCLUSION: LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.展开更多
Objective To construct lentivirus vectors carrying alphastatin gene,test its secretion expression in human umbilical vein endothelia cells(HUVECs)and observe its effects on growth,migration and tube formation of HUVEC...Objective To construct lentivirus vectors carrying alphastatin gene,test its secretion expression in human umbilical vein endothelia cells(HUVECs)and observe its effects on growth,migration and tube formation of HUVECs.Methods We constructed recombinant lentivirus vectors of NT4-alphastatin fusion gene containing neurotrophin-4 signal peptide,pro-region sequences and alphastatin,then transfected the recombinant lentivirus vectors into HUVECs to obtain secretory protein alphastatin and test its anti-angiogenic activities in vitro.Results Our data showed that recombinant self-inactivating lentivirus vectors of NT4-alphastatin were successfully constructed,and stable NT4-alphastatin transduced HUVECs were capable of sustainably secreting alphastatin which significantly suppressed HUVECs migration and differentiation but not VEGF-induced proliferation.Conclusion This report represents the first time on the use of lentivirus-based vectors to deliver alphastatin,the endogenous angiogenesis inhibitor,and reveals the potential utility of anti-angiogenic gene therapy with lentivirus vectors for treating cancer.展开更多
Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the su...Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the substantia nigra of rats were transfected with the Persephin gene. One week later 6-hydroxydopamine was injected into the anterior medial bundle to establish a Parkinson＇s disease model in the rats. Results found that the number of dopaminergic neurons in the substantia nigra increased, tyrosine hydroxylase expression was upregulated and concentrations of dopamine and its metabolites in corpus striatum were increased after pretreatment with Persephin gene. In addition, the rotating effect of the induced Parkinson＇s disease rats was much less in the group pretreated with the Persephin gene. Persephin has a neuroprotective effect on the 6-hydroxydopamine-induced Parkinson＇s disease through protecting dopaminergic neurons.展开更多
Smoothened （SMO） is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference,targeting the SMO gene （NM_005631） to observe its effect on S...Smoothened （SMO） is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference,targeting the SMO gene （NM_005631） to observe its effect on SMO expression,cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line,LNCaP,and in the androgen-independent prostate cancer cell line,PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors （pHelper1.0 and pHelper2.0） in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines,respectively. The expression level of SMO mRNA,tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction （qRT-PCR）,3-（4,5）-dimethylthiahiazo （-z-yl）-3,5-di-phenytetrazoliumromide （MTT） assay and flow eytometry,respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully.pGCSIL-GFP-723 had the highest interfering efficiency,named Lv-SIL-SMO723 after co-transfection,with which LNCaP and PC3 cell lines were infected. Compared with the control groups,results showed significantly decreased （P〈0.05） SMO mRNA expressions of LNCaP and PC3,lower mean percentage of S-phase cells and higher mean percentage of G_2/M phase cells,as well as obviously slow proliferation （P〈0.01） of LNCaP in the infected group. Yet,the proliferation of PC3 was not altered （P〉0.05）. In conclusion,the recombinant lentivirus particles were able to suppress SMO expression,regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.展开更多
AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HI...AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection(MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo.Immunohistochemistry staining revealed that 88.19%EGFP-positive trabecular meshwork(TM) cells were observed in the human anterior segment. Nevertheless,the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group(P 】0.05).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.展开更多
Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm（TE） lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic l...Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm（TE） lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA（shRNA） and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.展开更多
Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse...Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse from all known SIVs, including “SIVmac”, which infects captive rhesus monkeys. The novel SIVs are intriguingly homologous to HIV-1, based on serology and partial lentiviral genomic sequence analyses. Diverse lenti-viruses infect human and nonhuman primates (NHPs). There are more than 45 different “species-specific” simian immunodeficiency viruses (SIVs) that infect their cognate NHP hosts in natural habitats in Africa. Indian NHPs are not known to be infected by SIVs in the wild. Conventionally SIVs are named after their natural hosts, except for SIVmac, which infects captive rather than wild rhesus macaques. SIVmac is therefore a misnomer. It is a genetic variant of the African SIVsmm, which infects wild African sooty mangabey monkeys. SIVsmm is the progenitor of human immunodeficiency virus (HIV-2), while SIVcpz that infects wild chimpanzees is the progenitor of HIV-1. Although natural infections cannot be easily studied in wild NHP populations, we have previously reported co-infection of wild Indian NHPs by other retroviruses: simian retroviruses (SRVs) and Simian Foamy viruses (SFV). Apart from zoonosis, transmission of pathogens from humans to animals: anthroponosis, has also been reported in literature.展开更多
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 seq...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.展开更多
Schwann cell transplantation is considered one of the most promising cell-based therapy to repair injured spinal cord due to its unique growth-promoting and myelin-forming properties.A the Food and Drug Administration...Schwann cell transplantation is considered one of the most promising cell-based therapy to repair injured spinal cord due to its unique growth-promoting and myelin-forming properties.A the Food and Drug Administration-approved Phase I clinical trial has been conducted to evaluate the safety of transplanted human autologous Schwann cells to treat patients with spinal cord injury.A major challenge for Schwann cell transplantation is that grafted Schwann cells are confined within the lesion cavity,and they do not migrate into the host environment due to the inhibitory barrier formed by injury-induced glial scar,thus limiting axonal reentry into the host spinal cord.Here we introduce a combinatorial strategy by suppressing the inhibitory extracellular environment with injection of lentivirus-mediated transfection of chondroitinase ABC gene at the rostral and caudal borders of the lesion site and simultaneously leveraging the repair capacity of transplanted Schwann cells in adult rats following a mid-thoracic contusive spinal cord injury.We report that when the glial scar was degraded by chondroitinase ABC at the rostral and caudal lesion borders,Schwann cells migrated for considerable distances in both rostral and caudal directions.Such Schwann cell migration led to enhanced axonal regrowth,including the serotonergic and dopaminergic axons originating from supraspinal regions,and promoted recovery of locomotor and urinary bladder functions.Importantly,the Schwann cell survival and axonal regrowth persisted up to 6 months after the injury,even when treatment was delayed for 3 months to mimic chronic spinal cord injury.These findings collectively show promising evidence for a combinatorial strategy with chondroitinase ABC and Schwann cells in promoting remodeling and recovery of function following spinal cord injury.展开更多
Background In most colorectal carcinomas, the level of phospholipase C （PLC）-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the me...Background In most colorectal carcinomas, the level of phospholipase C （PLC）-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells. Methods Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction （RT-PCR） analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed. Results Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis （30%-40%） of LoVo cells.Conclusions PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.展开更多
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 ...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.展开更多
基金This work was supported by the Natural Science Foundation of Shanghai Municipality(No.03ZR14016).
文摘Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.
基金Supported by The Affiliated First People’s Hospital, Shanghai Jiao Tong University and the Board of Education Fund for Scientific Research of Shanghai, China, No. 06BE067
文摘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.
基金supported by grants from the National Basic Research Program of China (973 Program), No. 2012CB967900, 2012CB967904the National Natural Science Foundation of China, No. 81070782+2 种基金the Natural Science Foundation of Zhejiang Province, China, No. 30672308the Qianjiang Talent Project of Science and Technology Ministry in Zhejiang Province, No. 2011R10014the Natural Science Foundation of Ningbo, No. 2011A610042
文摘Lentivirus carrying the Atohl gene can infect Corti＇s organ and express a hair-like cell surface marker in the supporting cell area. However, expression of the gene carried by adenovirus is instantaneous, which undoubtedly limits its clinical application. Lentivirus acts as a carrier that can stably and continuously express genes. In this study, the cochlear structure and hearing level were not affected, and Atohl gene carried by lentivirus promoted the production of hair-like cells in the cochlear supporting cell area. This led to expression of the hair-like cell surface marker myosin 7a 30 days after lentivirus carrying Atohl was microinjected into the cochlear round window of rats.
基金Supported by the National Natural Science Foundation of China(No.81070735)
文摘AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated REDO gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.
基金Supported by the Natural Science Foundation of Yunnan Province(2002C0070M).
文摘Objective To explore the feasibility for therapy of spinal cord injury （SCI） by genetic engineering neural stem cell （NSC） modified by lentiviral vector. Methods Following the construction of the genetic engineering NSC modified by lentivirus to secrete both neurotrophic factor-3 （NT-3） and green fluorescence protein （GFP）, hemisection of spinal cord at the level of T10 was performed in 56 adult Wistar rats that were randomly divided into 4 groups （ n = 14 ）, namely 3 therapeutic groups and 1 control group. The therapeutic groups were dealed with NSC, genetic engineering NSC, and concentrated lentiviral supematant which carries both GFP and NT-3, respectively. Then used fluorescence microscope to detect the transgenic expression in vitro and in vivo, migration of the grafted cells in vivo, and used the Basso, Beattie, and Bresnahan （BBB） open-field locomotor test to assess the recovery of function. Results The transplanted cells could survive for long time in vivo and migrate for long distance. The stable transgenie expression could be detected in vivo. The hindlimb function of the injured rats in 3 therapeutic groups, especially those dealed with genetic engineering NSC, improved obviously. Concision It is feasible to combine NSC with lentivirus for the repair of SCI. NSC modified by lentivirus to deliver NT-3, acting as a source of neurotrophic factors and function cell in vivo, has the potential to participate in spinal cord repair.
基金supported by the Science and Technology Foundation Program of Jiangsu Province(Tumorigenic nucleostemin genes and adenovirus-based RNA interference targeting to brain tumor stem cell the rapy),No.BK2007072
文摘In the present study, we constructed a lentivirus, FIV-CMV-GFP-miR-7-3, containing the microRNA-7-3 gene and the green fluorescent protein gene, and used it to transfect human glioma U251 cells. Fluorescence microscopy showed that 80% of U251 cells expressed green fluorescence. Real-time reverse transcription PCR showed that microRNA-7-3 RNA expression in U251 cells was significantly increased. Proliferation was slowed in transfected U251 cells, and most cells were in the G1 phase of the cell cycle. In addition, the expression of the serine/threonine protein kinase 2 was decreased. Results suggested that transfection with a lentivirus carrying microRNA-7-3 can effectively suppress epidermal growth factor receptor pathway activity in U251 cells, arrest cell cycle transition from GI phase to S phase and inhibit glioma cell growth.
基金Supported by the Grants from the Project of Scientific Innovation and Creative for Jilin Provincial Oversea Scholars(No.2010273)Jilin Provincial Science and Technology Services(Nos.20050408, 20070720, 200805120, 20090732)+2 种基金Jilin Provincial Development and Reformation Committee(No.2009Y042J12314)Jilin Province Talent Development Foundation (No.JRJB2007-2)the Natural Science Foundation of China(Nos.30670301, 30870354)
文摘Cyclin-dependent kinase 2-associated protein 1（CDK2AP1）, a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, no studies on the function of CDK2AP1 in human lung cancer have been yet reported. In this study, overexpressing lentiviral vectors containing full-length CDK2AP1 cDNA and CDK2AP1 shRNA（short hairpin RNA） were constructed. Our results show that infecting A549 cells with lentivirus containing CDK2AP1 shRNA or full-length CDK2AP1 cDNA results in significantly down- or up-regulated the expression of CDK2AP1, respectively, thereby providing reliable tools for studying the function of CDK2AP1 in pulmonary carcinogenesis. Our data of MTT assay and flowcytometric analysis demonstrate that CDK2AP1 plays an important role in the proliferation/growth and cell cycling of A549 cells in vitro, and further investigation into its underlying mechanism of pulmonary carcinogenesis is needed.
文摘AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-xL and bcl-xS were detected with western blot. RESULTS: We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved indown-regulating bcl-xL and up-regulating bcl-xS. CONCLUSION: LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.
基金supported by the National Natural Science Foundation of China(No.30672162)
文摘Objective To construct lentivirus vectors carrying alphastatin gene,test its secretion expression in human umbilical vein endothelia cells(HUVECs)and observe its effects on growth,migration and tube formation of HUVECs.Methods We constructed recombinant lentivirus vectors of NT4-alphastatin fusion gene containing neurotrophin-4 signal peptide,pro-region sequences and alphastatin,then transfected the recombinant lentivirus vectors into HUVECs to obtain secretory protein alphastatin and test its anti-angiogenic activities in vitro.Results Our data showed that recombinant self-inactivating lentivirus vectors of NT4-alphastatin were successfully constructed,and stable NT4-alphastatin transduced HUVECs were capable of sustainably secreting alphastatin which significantly suppressed HUVECs migration and differentiation but not VEGF-induced proliferation.Conclusion This report represents the first time on the use of lentivirus-based vectors to deliver alphastatin,the endogenous angiogenesis inhibitor,and reveals the potential utility of anti-angiogenic gene therapy with lentivirus vectors for treating cancer.
基金supported by the National Natural Science Foundation of ChinaNo.81171208+1 种基金the Natural Science Foundation of Shandong Province of ChinaNo.Z2008C06
文摘Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the substantia nigra of rats were transfected with the Persephin gene. One week later 6-hydroxydopamine was injected into the anterior medial bundle to establish a Parkinson＇s disease model in the rats. Results found that the number of dopaminergic neurons in the substantia nigra increased, tyrosine hydroxylase expression was upregulated and concentrations of dopamine and its metabolites in corpus striatum were increased after pretreatment with Persephin gene. In addition, the rotating effect of the induced Parkinson＇s disease rats was much less in the group pretreated with the Persephin gene. Persephin has a neuroprotective effect on the 6-hydroxydopamine-induced Parkinson＇s disease through protecting dopaminergic neurons.
基金Acknowledgment This research was funded by National Natural Science Foundation of China （No. 30500506）.
文摘Smoothened （SMO） is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference,targeting the SMO gene （NM_005631） to observe its effect on SMO expression,cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line,LNCaP,and in the androgen-independent prostate cancer cell line,PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors （pHelper1.0 and pHelper2.0） in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines,respectively. The expression level of SMO mRNA,tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction （qRT-PCR）,3-（4,5）-dimethylthiahiazo （-z-yl）-3,5-di-phenytetrazoliumromide （MTT） assay and flow eytometry,respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully.pGCSIL-GFP-723 had the highest interfering efficiency,named Lv-SIL-SMO723 after co-transfection,with which LNCaP and PC3 cell lines were infected. Compared with the control groups,results showed significantly decreased （P〈0.05） SMO mRNA expressions of LNCaP and PC3,lower mean percentage of S-phase cells and higher mean percentage of G_2/M phase cells,as well as obviously slow proliferation （P〈0.01） of LNCaP in the infected group. Yet,the proliferation of PC3 was not altered （P〉0.05）. In conclusion,the recombinant lentivirus particles were able to suppress SMO expression,regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.
基金Supported by Natural Science Foundation of China(No.30901395)the Doctoral Fund of Ministry of Education of China(No.20090142120012,20110142120021)
文摘AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection(MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo.Immunohistochemistry staining revealed that 88.19%EGFP-positive trabecular meshwork(TM) cells were observed in the human anterior segment. Nevertheless,the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group(P 】0.05).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.
基金Supported by the Scientifi c Research Fund of Heilongjiang Provincial Education Department(11551039)
文摘Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm（TE） lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA（shRNA） and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.
文摘Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse from all known SIVs, including “SIVmac”, which infects captive rhesus monkeys. The novel SIVs are intriguingly homologous to HIV-1, based on serology and partial lentiviral genomic sequence analyses. Diverse lenti-viruses infect human and nonhuman primates (NHPs). There are more than 45 different “species-specific” simian immunodeficiency viruses (SIVs) that infect their cognate NHP hosts in natural habitats in Africa. Indian NHPs are not known to be infected by SIVs in the wild. Conventionally SIVs are named after their natural hosts, except for SIVmac, which infects captive rather than wild rhesus macaques. SIVmac is therefore a misnomer. It is a genetic variant of the African SIVsmm, which infects wild African sooty mangabey monkeys. SIVsmm is the progenitor of human immunodeficiency virus (HIV-2), while SIVcpz that infects wild chimpanzees is the progenitor of HIV-1. Although natural infections cannot be easily studied in wild NHP populations, we have previously reported co-infection of wild Indian NHPs by other retroviruses: simian retroviruses (SRVs) and Simian Foamy viruses (SFV). Apart from zoonosis, transmission of pathogens from humans to animals: anthroponosis, has also been reported in literature.
基金National Basic Research Program of China(No.2003CB515304)
文摘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.
基金supported in part by NIH R01 NS100531,R01 NS103481NIH R21NS130241(to LD)+3 种基金Merit Review Award I01 BX002356,I01 BX003705 from the U.S.Department of Veterans AffairsIndiana Spinal Cord and Brain Injury Research Foundation(No.19919)Mari Hulman George Endowment Funds(to XMX)Indiana Spinal Cord&Brain Injury Research Fund from ISDH(to NKL and LD)。
文摘Schwann cell transplantation is considered one of the most promising cell-based therapy to repair injured spinal cord due to its unique growth-promoting and myelin-forming properties.A the Food and Drug Administration-approved Phase I clinical trial has been conducted to evaluate the safety of transplanted human autologous Schwann cells to treat patients with spinal cord injury.A major challenge for Schwann cell transplantation is that grafted Schwann cells are confined within the lesion cavity,and they do not migrate into the host environment due to the inhibitory barrier formed by injury-induced glial scar,thus limiting axonal reentry into the host spinal cord.Here we introduce a combinatorial strategy by suppressing the inhibitory extracellular environment with injection of lentivirus-mediated transfection of chondroitinase ABC gene at the rostral and caudal borders of the lesion site and simultaneously leveraging the repair capacity of transplanted Schwann cells in adult rats following a mid-thoracic contusive spinal cord injury.We report that when the glial scar was degraded by chondroitinase ABC at the rostral and caudal lesion borders,Schwann cells migrated for considerable distances in both rostral and caudal directions.Such Schwann cell migration led to enhanced axonal regrowth,including the serotonergic and dopaminergic axons originating from supraspinal regions,and promoted recovery of locomotor and urinary bladder functions.Importantly,the Schwann cell survival and axonal regrowth persisted up to 6 months after the injury,even when treatment was delayed for 3 months to mimic chronic spinal cord injury.These findings collectively show promising evidence for a combinatorial strategy with chondroitinase ABC and Schwann cells in promoting remodeling and recovery of function following spinal cord injury.
基金This study was supported by the grants from National Natural Science Foundation of China(No.39870381)Natural Science Foundation of Guangdong Province(No.020015)Emphasis Natural Science Foundation of Guangdong Province(No.05102580).
文摘Background In most colorectal carcinomas, the level of phospholipase C （PLC）-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells. Methods Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction （RT-PCR） analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed. Results Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis （30%-40%） of LoVo cells.Conclusions PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.
文摘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.
