Construction of TLR4 shRNA plasmid and screening of human pancreatic cancer PANC1 cell line with stable transfection

Abstract

Objective To construct the eukaryotic plasmid expression vector mediated short hairpin RNA(shRNA) interference targeting TLR4 gene,and transfect it into pancreatic adenocarcinoma cell line PANC1,then screen stably transfected clonal cell line.Methods Three shRNA interference expression plasmid vectors targeting the TLR4 gene were constructed,named TLR4-1,TLR4-2,TLR4-3.The shRNA plasmid with highest inhibitory efficiency was selected and transfected into PANC1 cells with liposome.The silencing efficiency and transfection efficiency of TLR4-shRNA was assayed with real-time quantitative PCR and flow cytometry analysis.Monoclonal cell with stable transfection of TLR4-shRNA were selected by geneticin 418 (C418) and limiting dilution analysis.Results Transient transfection efficiency of PANC1 was (46.72 ±5.06) ％.TLR4 mRNA expressions were 0.025 ± 0.004,0.027 ± 0.003,0.019 ± 0.006in cells transfected with TLR4-1,TLR4-2,TLR4-3,respectively,which were significantly lower than that in untransfected group (0.061 ±0.018) and negative control group (0.057 ±0.015,P ＜0.05).The transfection efficiency of TLR4-3 vector in stably transfected clones [(82.79 ±8.16)％] was significantly higher than that of transient transfection (P =0.001 ).The expression of TLR4 mRNA was decreased to 0.010 ± 0.002,which was significantly lower than that of transient transfection ( P =0.001 ).The expression of TLR4 protein was (0.54±0.32) ％,which was significantly lower than that of untransfected cells [( 87.42 ± 5.00 ) ％] and that of negative control [(82.9±5.00)％,P =0.000].Conclusions Stable transfection PANC1 cell lines with TLR4 gene silencing are successfully identified. Key words: Pancreatic neoplasms; Toll like receptor4; Transfection; RNA interference