| Background & Objective The growth and metastasis of tumor tissues depend on formation of new vascular and vascular endothelial growth factor 165(VEGF 165),highly expressed in most tumor tissues and cell lines,is important angiogenesis promoter.Although neuropilin-1(NRP-1) was initially deseribed as a mediator of neuronal guidance,it appears to be a mediator of angiogenesis.NRP-1 expressed in endothelial cells enhances VEGF 165 binding to VEGFR2 and then plays important roles in modulating growth and metastasis of kinds of tumors.We aim to inhibit expression of VEGF 165 and NRP-1 in primary liver cancer cell lines HCCLM6 by lentivirus-mediated RNA interference(RNAi) and investigate its effect on primary liver cancer' growth and lung matastasis in vitro and vivo.Methods Screen out the efficient and safe RNAi vector by flow cytometer detecting efficiencies of lentivirus,liposome and magnetic nanoparticle in carrying non-specific short hairpin RNA(shRNA);VEGF165 shRNA expression plasmid and NRP-1shRNA expression plasmid were separately constructed and identified using DNA recombinant technology.Constructed plasmid and lentivirus elements were cotransfected into 293T cells to pack up mature lentiviruses.Then HCCLM6 cells were infected with two mature lentiviruses separately and combinely.48 hours later,efficiencies of different lentiviruses were observed by flow cytometer,VEGF 165 and NRP-1 mRNA expression were measured by real time PCR,VEGF 165 and NRP-1 protein expression were detected by western blot and HCCLM6 cells' growth alteration were measured by MTT method.Different groups cells were inculated into nude mice to establish cancer xenograft.Tumor growth and lung matastasis were monitored.The expression of VEGF 165 and NRP-1 protein in tumor tissues were detected by Western blot.Microvessel density(MVD) in tumor tissues was detected by immunohistochemistry.Results 1 Transfection efficiency of lentiviruses was highest(90.1%) followed by liposomes(60.8%).Magnetic nanoparticles were the least efficient vector(30.2%). Compared to the wild type strain,cells transfected with lentiviruses and liposomes grew more slowly without statistically significance(P＞0.05).Little variance was found between cells transfected with magnetic nanoparticles and wild type strains(P＞0.05);2 All the positived clones were right clones and the two mature lentiviruses'titer was the same(5×10~8/ml);3 The efficiencies of VEGF 165 shRNA,NRP-1shRNA and co-shRNAs were 86.8%, 97.7%and 99%;Compared to the wild type strain,VEGF165 mRNA expression was inhibited by 65%in cells transfected with the VEGF 165shRNA,70%in cells transfected with co-shRNAs.Little variance was found in cells transfected with NRP-1shRNA and naked vector.NRP-1 mRNA expression was inhibited by 81%in cells transfected with the NRP-1shRNA,80%in cells transfected with co-shRNAs.Little variance was found in cells transfected with VEGF 165shRNA and naked vector;Compared to the wild type strain, VEGF165 protein expression was inhibited by 57.1%in cells transfected with the VEGF 165shRNA,57.2%in cells transfected with co-shRNAs(P＜0.05).Little variance was found in cells transfected with NRP-1shRNA and naked vector(P＞0.05).NRP-1 protein expression was inhibited by 74.9%in cells transfected with the NRP-1shRNA,74.6%in cells transfected with co-shRNAs(P＜0.05).Little variance was found in cells transfected with VEGF 165shRNA and naked vector(P＞0.05).The cell growth curve indicated,cells transfected with VEGF 165shRNA,NRP-1shRNA and co-shRNAs grew more slowly than the wild type strain,especially for the cells transfeted with co-shRNAs(P＜0.05).Little changes was found between cells with naked vector and wild type(P＞0.05).4 The tumor size and weight of VEGF 165shRNA transfected group,NRP-1shRNA transfected group and co-shRNA transfected group were significantly reduced,comparing to control group (P＜0.05).Little variance was found in naked vector transfeced group and control group (P＞0.05).6 nude mice in control goup and naked vector transfected group all occurred lung matastasis,4 nude mice in VEGF165shRNA transfected group and NRP-1shRNA transfected group occurred lung matastasis,3 nude mice in co-shRNA transfected group occurred lung matastasis.VEGF165 protein expression of tumor tissues was inhibited by 56%in VEGF 165shRNA transfected group,56.1%in co-shRNAs transfected group (P＜0.05).Little variance was found in NRP-1 shRNA transfected group and naked vector transfected group(P＞0.05).NRP-1 protein expression was inhibited by 52.2%in NRP-1shRNA transfected group,54.1%in co-shRNAs transfected group(P＜0.05).Little variance was found VEGF 165shRNA transfected group and naked vector transfected group (P＞0.05).MVD of VEGF 165shRNA transfected group,NRP-1shRNA transfected group and co-shRNAs transfected group were significantly reduced,comparing to control group (P＜0.05).Little variance was found in naked vector transfeced group and control group (P＞0.05).Conclusion The gene expression of VEGF165 and NRP-1 was significantly inhibited in cells transfected with co-shRNAs,whose growth was also suppressed.In vivo,MVD,growth and lung matastasis of nude mice tumor were suppressed by co-shRNAs.
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