Effect Of Lentivirus-induced SiRNA Targeted Against Toll-like Receptor 3 On The Proliferation And Immune Escape Of Human Pulmonary Adenocarcinoma Cells Line A549

Partâ… : The expression and significance of TLR₃ in humanpulmonary adenocarcinoma cells line A549Objective : Being considered as a bridge between the innate immunity and acquiredimmunity, Toll-like receptors are very important innate immunity moleculars. Recentresearches on the innate immunity have focused on the relationship between Toll-likereceptors and human tumor, This paper investgates the expression and significance ofTLR₃ in human pulmonary adenocarcinoma cell (A549 cell) and human bronchialepithelial cell (HBE cell).Methods: After culturing A549 cell and HBE cell in vitro, the expression of TLR₃mRNA and protein in both cells were detected by immunocytochemistry, real-timequantitative reverse transcriptase-polymerase chain reaction (Real-Time Quantitative PCR)and western blot, respectively.Results: By immunocytochemistry staining, TLR₃ is mainly expressed in both cells'cell membrane and endochylema as brown-yellow material. It shows that the expressions of TLR₃ mRNA and protein in A549 cell are stronger than those in HBE cell (p＜0.01).Conclusion: The resusts suggest TLR₃ might cause the progression ofhuman pulmonary adenocarcinoma, and the mechanism needs to be furtherinvestgated.Partâ…¡: Construct lentiviral RNAi vectors targeted againstToll-like receptor 3 geneObjective : To observe the influence of lentiviral vector-mediated RNA interferenceon expression of human TLR3(Toll-like receptor 3) gene in human pulmonaryadenocarcinoma cell line A549, so as to pave a way for TLR3 gene-targeted gene therapyof pulmonary adenocarcinoma.Methods: Gene engineering technique was used to screen four RNA interferencesequences targeting TLR3 gene; the sequences were separately cloned into the pCCL-GFPvector to construct TLR3-RNAi-LV 1# , TLR3-RNAi-LV 2# , TLR3-RNAi-LV 3#,TLR3-RNAi-LV 4#, which were subsequently confirmed by PCR and DNA sequencinganalysis. The titer of lentivirus was determined after 293T cells were contransfected withTLR3-RNAi-LV,pHelper 1.0,pHelper 2.0. The four kinds of recombinant lentiviruseswere injected into A549 cells and the TLR3 mRNA and protein expression were examinedby real-time RT-PCR and Western blotting, respectively, and the result was compared withthose of the non-transfected and blank vector transfected A549 cells.Results: PCR analysis and DNA sequencing confirmed that the four TLR3 vshRNAsequences were successfully inserted into the lentiviral vectors. The titer of concentratedvirus were 2E+8,2E+8,2E+8,1E+8 TU/ml ,respectively. TLR3 expression in A549 cellswas significantly inhibited at both mRNA and protein levels compared with thenon-tansfected and empty vector transfected A549 cells.(P＜0.05) After transfection withTLR3-RNAi-LV 4#, TLR3 mRNA expression decreased by 80ï¼…, TLR3 protein expression decreased by 68ï¼…. (P＜0.05)Conclusion: Four lentiviral RNAi vectors of TLR3 gene have been successfullyconstructed, and they can effectively inhibit the expression of TLR3 gene in A549 cellsin vitro.Partâ…¢: Effect of lentivirus-induced siRNA targeted againstToll-like receptor 3 on the proliferation and immune escape ofhuman pulmonary adenocarcinoma cells line A549Objective: To evalute the effect of Toll-like receptor 3 on the cell proliferate,anti-apoptotic and immune escape in human pulmonary adenocarcinoma cells line A549with different expressions of Toll-like receptor 3.Methods: To transfecte A549 cell line with TLR3-RNAi lentiviral vector in thesecond experiment, and transfected,non-transfected and blank vector transfected A549cells were all incubated with Poly(I:C),the ligand of TLR3. The expressions of B7-H1,PGE-2 and phosphorylase p38 were respectively evaluated by flow cytometry,real-timequantitative PCR and western blot experiments. Apoptosis of those cells was detected byflow cytometry with AnnexinV FITC/PI double staining.Results: Under the condition of the same time and concentration of stimulation withPoly(I:C), the percentage of B7-H1-positive cells of TLR3-RNAi lentiviral vectortransfected A549 cells is 23.35±0.071ï¼…, those of the non-transfected and blank vectortransfected A549 cells were 55.25±0.015ï¼…,48.75±0.020ï¼…, respectively。Theexpression of B7-H1 of TLR3-RNAi lentiviral vector transfected A549 cells wassignificantly lower than those of the non-transfected and blank vector transfected A549cells (p＜0.01); To analyze with the 2-ΔΔCt method, The expression of PGF-2 ofTLR3-RNAi lentiviral vector transfected A549 cells was significantly lower than thoseof the non-transfected and blank vector transfected A549 cells (p＜0.01); the relativelypercentage of phosphorylase p38 of TLR3-RNAi lentiviral vector transfected A549 cellsis 6.37±0.28ï¼…those of the non-transfected and blank vector transfected A549 cells were 33.63±6.752ï¼…,17.36±2.24ï¼…, respectively。The expression of phosphorylase p38of TLR3-RNAi lentiviral vector transfected A549 cells was significantly lower thanthose of the non-transfected and blank vector transfected A549 cells (p＜0.01); Thepercentage of the apoptosis cells of TLR3-RNAi lentiviral vector transfected A549 cellsis 44.4±1.41ï¼…, those of the non-transfected and blank vector transfected A549 cellswere 34.7±0.89ï¼…,36.5±1.12ï¼…, respectively。The percentage of the apoptosis cells ofTLR3-RNAi lentiviral vector transfected A549 cells was significantly higher than thoseof the non-transfected and blank vector transfected A549 cells (p＜0.01 )Conclusions: Down-regulation of TLR3 led to inhibit the expression ofB7-H1,PGE-2 and phosphorylase p38 in human pulmonary adenocarcinoma cells lineA549, weaken the anti-apoptosis ability of human pulmonary adenocarcinoma cells lineA549.