Study On The Molecular Mechanism Underlying The Process Of Bone Metastasis By Lentivirus Mediated ShRNA Targeting Idl Gene In A549Lung Cancer

Objective:To construct a lentiviral vector with shRNA targeting Idl gene for transfection.Methods:(1)lentivirus vector pFU-GW-RNAi-GFP plasmid was digested Hpa I and XhoI enzymes. Then, a pair of nucleotide oligonucleotides encoding shRNA targeting to Id1gene were annealed and cloned into the Hpa I and XhoI sites of the pFU-GW-RNAi-GFP lentivirus vector. The resulting vector, designated pFU-GW-ID1shRNA-GFP, was subsequently verified by sequencing.(2)lentiviral particles were generated by transfecting pFU-GW-shRNA-ID1-GFP lentivirus vector, plasmids pHelper1.0and pHelper2.0into293T cells using Lipofectamine2000. The lentiviral particles (Lenti-Idl shRNA) were generated and concentrated to store.The titer was determined.Results:DNA sequencing verified that the sequence of shRNA targeting Idl gene was successfully cloned into pFU-GW-RNAi-GFP lentivirus vector and titer of Lenti-Idl shRNA was2x109Tu/ml.conclusion:Lentiviral vector with shRNA targeting Idl gene(pFU-GW-ID1shRNA-GFP) was ssuccessfully constructed. Objective:To explore the role of Idl gene in the growth and invsion process of A549cell and its effect to expression of TSP-1Methods:A549cells were transfected by high titer lentiviral particles exprssing Id1shRNA (Lenti-Id1shRNA) or negative control shRNA (Lenti-NCshRNA).(1)The transfection efficiency was assessed though the observation of GFP by flurorescent micrscope.(2)Id1gene RNAi efficiency was determined though measuring Idl protein lever by western blot.(3) Cell proliferation, cell cycle and cellinvasion were assessed by CCK8,PI staining and transwell assay.(4)Realtime PCR was used to determine the Idl and TSP-1mRNA expression levers.Results:96h after the cells were transfected with lentiviral particles, bright green fluorescence in the A549cell was observed under the fluorecent microscope,the best multiplicity of infection(MOI) were20,and the transfection efficiency reach about96%. Idl protein in Idl shRNA group was lower than the NCshRNA group(p<0.05). Cell proliferation in IdlshRNA group decreased significantly comparing with the control group(p<0.001). cell cycle anaysis showed that G1phase cell fraction in Id1shRNA group increased significantly comparing with the control group (85.10±1.31%vs.60.49±0.12%, P <0.05);but S phase cell fraction in Id1shRNA group decreased significantly(7.32±0.99%vs.35.55±0.15%, P<0.001). Cell invasion in Id1shRNA group reduced by about50%(P<0.05). TSP-1mRNA in IdlshRNA group were about6.87times higher than the control group(p<0.05).conclusion:lentiviral delivery of Idl shRNA resulted in effieicent Idl gene silencing. Idl gene silencing in A549cells led to reduced cell proliferation,cell cycle G1-S phase block and reduced cell invasion. Idl gene silencing promoted TSP-1mRNA expression in A549cells Objective:To construct bone metastatic nude mice models with A549tumor cells and to explore the role of Id1in bone metastasis process.Methods:43male Balb/c nude mice of4-5weeks old were purchased and randomly divided in to three group:Id1shRNA group(n=20), NCshRNA group(n=20) and blank group (n=3). A549cells were transfected by lentiviral particles Lenti-Id1shRNA or Lenti-NCshRNA,96h later, the cells were collected in PBS. In Id1shRNA or NCshRNA group, A549cells(5x105cells in30u1PBS each mouse) were injected into medullar cavity of tibias of mice wth26G needles. In blank group,30u1PBS without A549cells was injected.Then,the mice were kept for3or6weeks. X-ray and histologic check were performed to examine bone lesion caused by tumor. Osteoclasts in sections were determined by Tartrate-resistant acid phosphate (TRAP) staining.Results:2mice died in Id1shRNA group and NCshRNA group. Radiography at3weeks showed that50%(5/10)mice in Id1shRNA group and10%(1/10)mice in NCshRNA group formed osteolytic lesion in the tibias of mice; Radiography at6weeks showed that87.5%(7/8)mice in IdlshRNA group and250%(2/8) mice in NCshRNA group formed osteolytic lesion.The osteolytic lesion in Id1shRNA group was much serious than the NCshRNA group. Histologic analysis showed that in Id1shRNA group there were larger tumor area and more tumor numble in the bone compared to NCshRNA group. There were cortical obliteration caused by tumor in ID1shRNA A549group but much less or no cortical erosion in NCshRNA group. TRAP staining indicated that osteoclasts at the bone-tumor interface in the ID1shRNA group are much more than NCshRNA group.conclusion:bone metastatic nude mice models were successfully constructed by injecting A549cells into medullar cavity of tibias. lentivirus mediated Idl gene silencing result in much more serious osteolytic lesion caused by A549lung cancer.Idl gene may inhibit cancer metastasis to bone.