Recombinant Lentivirus Containing CD36 Short Hairpin RNA Inhibits Activation Of L-TGF-β1 Derived From Rat Alveolar Macrophage And Silicosis In Rat

PrefaceIn China,pneumoconiosis is one of the most serious occupational diseases which are harming workers' health.Until the end of 2006,there are totally 616442 cases of pneumoconiosis,and it increases 8000 to 9000 every year.Though the government has already took a lot of measures,such as controlling the production and diffusion of dust, decreasing concentration of dust,improving operational environment,which had got some achievements.Because the harm of dust is also rigorous,preventing and controlling of pneumoconiosis are also important.It is difficult to cure the pathological changes of pneumoconiosis,but lung fibrosis is chronic and progressive pathology reactivity.To discuss the molecular mechanism of lung fibrosis also has practical significance for delaying and inhibiting the occurrence and the development of fibrosis.Investigations suggested that transforming growth factor-β1(TGF-β1)played a critical role in the occurrence and the development of the lung fibrosis.After lung is injured,alveolar macrophage is activated,and it generates much TGF-β1.TGF-β1 is binded with its receptor on membrane of lung fibroblast,and then the smads signal transduction pathway of intracellular has been activated.After the signal gets into nucleus,transcription proteins in nuclear are activated,and the mRNA transcription of collagen protein is started.Then the collagen protein is synthesized,and its degradation is inhibited.Finally lung fibrosis is formed.However,only activated TGF-β1 has bioactivity by binding with its receptor.Usually,activated alveolar macrophages secrete a lot of latent TGF-β1(L-TGF-β1).It hasn't bioactivity because it can't interact with its receptor.However,activated alveolar macrophages also secrete a lot of thrombospondin-1(TSP-1)and plasmin.TSP-1 can interact with L-TGF-β1 to form TSP-1/L-TGF-β1 complex,and the configuration of TSP-1/L-TGF-β1 complex has changed by binding with CD36 that is the receptor of TSP-1 on the membrane of alveolar macrophages.At this time,the plasmin nearby can make the latent TGF-β1 from the latent precursor to the biologically active form.RNA interference(RNAi)is a kind of reliable gene silencing technique,which could suppress the expression of target gene by using 21-23 nt double small interfering RNA(siRNA)or short hairpin RNA(shRNA).Lentivirus vector is a kind of hopeful transgenic vector which can infect non-dividing cells.The exogenous gene can achieve a sustained expression by the stable integration of lentivirus vector into host cell genome.So the lentvirus-mediated RNA interference will make it possible to silence the target gene persistently.Accordingly,CD36 plays an important role during the process of L-TGF-β1 activation.In the present study,we chose CD36 as our targeting gene,used RNAi technology to construct recombinant lentivirus with rat CD36 short hairpin RNA.We used it into the experiment in vitro and vivo,to observe the inhibition of L-TGF-β1 activation and silicosis,which may lay a foundation for studies on the molecular mechanism of silicosis.Methods1.Construction and identification of rat CD36 shRNA recombinant plasmidsAccording the sequence of rat CD36 mRNA in the Genbank,four targeting sequences and a negative sequence were designed.The synthesized sense and antisense oligo nucleotides were annealed.Then the double-strand oligo-DNA was linked to the linear plasmid pGCL-GFP.The production of recombinant plasmids afterwards transformed the competence E.coli DH5α.To identify the positive clone,single positive clone was then chosen.After PCR and DNA sequencing confirmation,the recombinant plasmids were named as pGCL-GFP-CD36-1,pGCL-GFP-CD36-2, pGCL-GFP-CD36-3,pGCL-GFP-CD36-4 and pGCL-GFP-CD36-NC.2.Construction and identification of eukaryotic expression vector containing CD36 of ratCD36 gene was amplified by RT-PCR from total RNA of normal rat alveolar macrophage NR8383.The reconstructed expression vectors were constructed by enzyme digestion and cloning into eukaryotic expression vector pEGFP-N1.After restriction endonuclease digestion,PCR and DNA sequencing confirmation,the recombinant CD36 expression plasmid was named as pEGFP-N1-CD36.3.Efficiency analysis of pGCL-GFP-CD36 shRNA plasmids by western blotpGCL-GFP-CD36-(1,2,3,4,NC)together with pEGFP-N1-CD36 co-transfected 293T cells under Lipofectamine 2000,respectively.Forty-eight hours after co-transfection,the cells were harvested and total proteins were extracted for western analysis.Western blot assay was performed for selecting the targets that could interference the expression of CD36.4.Production of recombinant lentiviruses and titrationLentiviruses were produced in 293T cells by transient transfection of three plasmids:transfer vector pGCL-GFP-CD36-(1,2,3,4,NC),the packaging vector pCMV-dR8.74 and the VSV-G expression plasmid pMD2G.The medium was replaced 8 h later,and virus particles released into the medium were harvested 40 h after transfection.The recombinant lentiviruses were named as Lv-shCD36-1,Lv-shCD36-2, Lv-shCD36-3,Lv-shCD36-4 and Lv-shCD36-NC.The virus medium was purified by ultracentrifugation,and viral titration was measured by checking for GFP fluorescence under fluorescence microscope.5.Efficiency analysis of Lv-shCD36 in NR8383 cells by realtime-PCR and western blotLentviruses viral supernatants were incubated with NR8383 cells for 8 h,and then changed the medium.After 40 h incubation,GFP fluorescence was checked under fluorescence microscope.If the efficiency of infection is lager than 50%,then incubated for another 48 h,collected the cells for realtime-PCR and western blot assay.6.Inhibition of L-TGF-β1 activation in NR8383 cells by Lv-shCD36The experiment was divided into three groups,which were bleomycin(BLM) group,BLM+Lv-shCD36 group,and BLM+Lv-shCD36-NC group.Firstly,we used NR8383 cells infected with Lv-shCD36 and NR8383 cells infected with Lv-shCD36-NC to plate at a density of 1×10⁶ cells/ml in serum-free F12K in 6-well plates.After 2 h at 37℃,cells were given serum-free F12K medium containing 0.1μg/ml concentration of bleomycin,and incubated for 18 h.The cells of each group were changed the medium and incubated for 24 h.And then the cells were collected for detecting the interference effect by real-time PCR and western blot,and the supernatant were collected for TGF-β1 by CCL-64 mink lung epithelial growth inhibition assay.7.Inhibition of L-TGF-β1 activation and silicosis in rat by Lv-shCD36Animals were divided randomly into the following four experimental groups(n= 24 per group):(1)saline control group:instilling 0.5 ml sterile physiological saline;(2) SiO₂ group:instilling a suspension of 10mg SiO₂ in a total volume of 0.5 ml sterile physiological saline;(3)SiO₂+Lv-shCD36 group:instilling a mixed suspension of 10mg SiO₂ and 5×10⁸ TU Lv-shCD36 in a total volume of 0.5ml sterile physiological saline;(4)SiO₂+Lv-shCD36-NC group:instilling a mixed suspension of 10mg SiO₂ and 5×10⁸ TU Lv-shCD36-NC in a total volume of 0.5ml sterile physiological saline. At 7,21 and 28 days post-instillation,8 rats of each group were anesthetized with anesthetic ether,sacrificed by decapitation,and the lungs and hilar lymph nodes were removed.The alveolar lavage fluid was collected for the activation of TGF-β1 assay, and the alveolar macrophages(AMs)were collected for checking GFP fluorescence under fluorescence microscope.The left lobe of lung was fixed with 4% paraformaldehyde,embedded in paraffin,and sectioned at 5μm The tissue sections were stained with hematoxylin and eosin(HE)and van Gieson stains(vG).The right lobe of lung was used to determine the hydroxyproline content.And immunohistochernical examination of collagenâ… andâ…¢was performed.8.Statistical analysisResults were expressed as mean±S.E.M.All analyses were carried out using the SPSS 13.0 software.Differences among multiple groups were analyzed using one-way analysis of variance(ANOVA)followed by Student-Newman-Keuls(SNK)test when F was significant.P values of less than 0.05 were considered statistically significant.Results1.Construction of recombinant plasmids pGCL-GFP-CD36The results of PCR and DNA sequencing showed that we had successfully constructed the recombinant plasmids pGCL-GFP-CD36-1,-2,-3,-4,-NC.2.Construction of recombinant plasmid pEGFP-N1-CD36The results of PCR,enzyme digestion and DNA sequencing showed that we had successfully constructed the recombinant plasmid pEGFP-N1-CD36.3.CD36 silencing effect detection of pGCL-GFP-CD36 in 293T cellsGFP expression was observed with fluorescent microscope 48 h after pGCL-GFP-CD36-1,-2,-3,-4,-NC plamids co-transfected with pEGFP-N1-CD36 in HEK-293T cells respectively.Results of western blot showed that 0.25μg and 0.5μg pGCL-GFP-CD36-1,-2,-3,-4 could significantly inhibit 0.5μg pEGFP-N1-CD36 plasmids expression in HEK-293T cells,while pGCL-GFP-CD36-NC did not influence the expression of the pEGFP-N1-CD36 plasmid in HEK-293T cells.4.Results of recombinant lentiviruses titrationThe infectious titers of Lv-shCD36-1,-2,-3,-4,-NC were determined by GFP assay under a fluorescent microscope.Fluorescent cells were reduced with the multiple of dilution increased.We counted the number of fluorescent cells at the hole which had 10%fluorescent cells,then multiplied the value with the corresponding dilution to obtain the titrations of recombinant lentiviruses.The titrations of Lv-shCD36-1,-2,-3, -4 were 2×10⁹ TU/ml medium,and the titration of Lv-shCD36-NC was 3×10¹⁰TU/ml medium.5.Detection of CD36 silencing effect by Lv-shCD36 in NR8383 cellsNR8383 cells were respectively infected with Lv-shCD36-1,-2,-3,-4,-NC.Then we performed real time-PCR to determine the mRNA level of CD36 in NR8383 cells. The results of real time-PCR showed that the infection of Lv-shCD36 -2,-3,-4 could suppress the CD36 mRNA expression for about 58%,73%,80%compared with infection of Lv-shCD36-NC,respectively.It demonstrated that Lv-shCD36-2,-3,-4 could significantly suppress the CD36 mRNA expression in the NR8383 cells.And the result of western blot showed that the infection of Lv-shCD36-2,-3,-4 could suppress the CD36 protein expression in NR8383 cells compared with Lv-shCD36-NC. According to the results of real-time PCR and western blot assay,the most obvious gene silencing effect was observed when Lv-shCD36-4 was applied.Thus Lv-shCD36-4 was used as Lv-shCD36 in the following research. 6.Inhibition of L-TGF-β1 activation in NR8383 cells by Lv-shCD36The results of real time-PCR and western blot assay showed that Lv-shCD36 could significantly suppress the CD36 mRNA and protein expression after the cells activated by bleomycin.In the CCL-64 growth inhibition assay,active TGF-β1 and percent of active TGF-β1 in the BLM+Lv-shCD36 group were significantly lower than those in the BLM group and BLM+Lv-shCD36-NC group(P＜0.05).7.Inhibition of L-TGF-β1 activation in BALF and silicosis of rat by Lv-shCD36The result of fluorescence assay showed that the AMs of Lv-shCD36 and Lv-shCD36-NC groups were able to observe the expression of fluorescent,which suggested the AMs were infected with Lv-shCD36 and Lv-shCD36-NC successfully. The result of real time-PCR showed that expression of CD36 mRNA in the Lv-shCD36 group was significantly lower than that of the saline control group,SiO₂ group and SiO₂+Lv-shCD36-NC group(P＜0.05)at 7 days after the instillation.The result of CCL-64 growth inhibition assay showed that the percent of active TGF-β1 of BALF in the SiO₂+Lv-shCD36 group was significantly lower than that of SiO₂ group and SiO₂+Lv-shCD36-NC group(P＜0.05)at 7 days after the instillation.Lung coefficient and hydroxyproline content of SiO₂+Lv-shCD36 group were significantly lower than SiO₂ group and SiO₂+Lv-shCD36-NC group(P＜0.05)at 21 and 28 days after the instillation.The result of pathologic examination showed that the degree of lung fibrosis in SiO₂+Lv-shCD36 group was significantly lighter than SiO₂ group and SiO₂+Lv-shCD36-NC group at 21 and 28 days after the instillation.The expression of collagenâ… andâ…¢in SiO₂+Lv-shCD36 group were significantly lower than SiO₂ group and SiO₂+Lv-shCD36-NC group(P＜0.05).Conclusion1.We construct a lentviral vector expressing the short hairpin RNA(shRNA)that is specific to CD36 of rat successfully,named Lv-shCD36.It is demonstrated that Lv-shCD36 could suppress the CD36 expression of NR8383 cells.2.Lv-shCD36 can inhibit the activation of L-TGF-β1 derived from bleomycin-treated NR8383 cells.3.Lv-shCD36 can infect the AMs in silicosis model of rat successfully,and it can surppress the expression of CD36.Lv-shCD36 can inhibit the activation of L-TGF-β1 of BALF and the development of silicosis.