| AIM:To investigate the expression of Toll-like receptor (TLR) 4, nuclear factor-kappaB(NF-κB) p65 and hypoxia-inducible transcription factor 1α(HIF-1α) in pancreatic ductal adenocarcinoma.METHODS:TLR4 mRNA and HIF-1αmRNA were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues, and expression of TLR4, NF-κB p65 and HIF-1αprotein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent tissues. The relationship between TLR4 or HIF-1αand pathologic features, association between TLR4 and HIF-1α, were also analyzed. Kaplan-Meier Method was used to assess the impact of TLR4 and HIF-1αexpression on survival of patients.RESULTS:The relative quantification of TLR4 mRNA and HIF-1αmRNA in tumor tissues were 0.81±0.10 and 0.87±0.11 respectively, which were significantly higher than that in adjacent tissues (0.70±0.16 and 0.68±0.13 respectively, P<0.05). The protein expression of TLR4, NF-κB p65 and HIF-1αin tumor tissues were 69.20%,66.15% and 70.80% respectively, significantly higher than that in adjacent normal tissues(39.50%, 31.58% and 36.80%, respectively, P< 0.05). There were no significant correlation between TLR4 or HIF-1αexpression and the age, gender, tumor location, the degree of tumor differentiation in patients (P>0.05). However, there were significant correlation between the expression of TLR4 or HIF-1αand tumor size, lymphnode metastasis, venous invasion and clinical staging(P<0.05). The expression of TLR4 and HIF-1αhad a significant impact on survival of patients with pancreatic adenocarcinoma.CONCLUSION:TLR4, NF-κB p65 and HIF-1αare overexpressed in pancreatic adenocarcinoma, TLR4 may be partly involved in up-regulating HIF-1α, and both synergyly accelerate progresis of pancreatic adenocarcinoma.AIM:To investigate the effects of eukaryotic plasmic expression vector mediated short hairpin RNA interference targeting TLR4 gene on pancreatic adenocarcinoma cells.METHODS:We constructed three short hairpin RNA (shRNA) interference expression plasmid vectors targeting the TLR4 gene and a control vector containing random DNA fragment.The recombinant plasmids were amplified in E.coli. DH5 a was identified by restriction digestion, PCR and sequencing. The vevtors were tansfected into PANC-1 cells. TLR4 mRNA was detected by real-time PCR, the most effective vector TLR4 RNAi-3# was transfected into PANC-1 and stably transfected clones was obtained by Geneticin (G418) and limiting dilution analysis. The transfection efficiency of plasmids were detected through evaluation of GFP by flow cytometry analysis, and the silencing effect of TLR4 was assayed with real-time quantitative PCR and Flow cytometry analysis.RESULTS:The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments. At 48 h after transfection, the transfection efficiency of plasmids was 46.72%±5.06%, and TLR4 mRNA was significantly down-regulated, especially in cells transfected with TLR4 RNAi-3# vector (0.019±0.006 vs 0.061±0.018, P=0.000). The transfection efficiency of TLR4 RNAi-3# vector in stably transfected clones was significantly higher than that of transient transfection (82.79%±8.16%vs 46.72%±5.06%, P=0.001). TLR4 mRNA was absolutely silenced (0.010±0.002 vs 0.019±0.006, P=0.01). The expression of TLR4 protein on stably transfected clones was significantly lowed than that on PANC-1 cells or cells transfected with control plasmid(0.54%±0.32% vs 87.42%±5%; 0.54%±0.32% vs 82.9%±5%, P=0.000).CONCLUSION:Plasmid vector expressing shRNA against TLR4 has been successfully constructed and it can down-regulated TLR4 on pancreatic adenocarcinoma effctively.AIM:To explore the role of the crosstalk of TLR4 and HIF-1αin pancreatic ductal adenocarcinoma and the possible mechanism.METHODS:The expression of TLR4 and HIF-1αprotein were detected by immunohistochemistry in pancreatic ductal adenocarcinoma and its adjacent tissues. Then the expression of HIF-1αwas observed in the human pancreatic cancer cell line PANC-1 by real time PCR and Western blotting after treated with cobalt chloride (CoC12, a hypoxia mimicking agent), LPS and PDTC(NF-kappa B inhibitor) respectively. RNA interference expression vector for TLR4 was transfected into PANC-1 cells, and the stably transfected clones were treated with CoC12 and LPS, then the expression of HIF-la was detected by real time PCR and Western blotting, and the NF-kappa B p65 nuclear translocation in was analyzed using immunofluorescence staining.RESULTS:The protein expression of TLR4 and HIF-la in tumor tissues were 69.20% and 70.80% respectively, significantly higher than that in adjacent normal tissues respectively (69.2% v.s 39.5%, P=0.003;70.8% v.s 36.8%, P=0.001). HIF-1αexpression was positive correlation with TLR4 (rp=0.451, p<0.05). LPS and CoC12 significantly increase HIF-la mRNA and protein in PANC-1 cells compared with CoC12 alone, both silencing of TLR4 and a specific NF-κB inhibitor, PDTC, suppressed the increase of HIF-la induced by LPS. Nuclear translocation of NF-κB p65 was induced by LPS in PANC-1 cells.CONCLUSION:TLR4 and HIF-1αare overexpressed in pancreatic adenocarcinoma TLR4 signaling regulates HIF-la mRNA and protein expression via NF-κB activation in pancreatic tumor microenviroment.AIM:To investigate the role of TLR4 signal pathway in adhesion and invasion of human pancreatic cancer cells to cell-extracellular matrix.METHODS:PANC-1 cells, PANC-1 RNAi Ctro cells and PANC-1 RNAi TLR4 cells were stimulated with LPS respectively, in vitro tumour cell ECM adhesion and ECM invasion were analysed by ECM adhesion assay and ECM invasion chambers. The expression of Integrinβ1 and their alteration by LPS were examined by flow-cytometric analysis, and the level of MMP-9 in cellular supernatant was detected by enzyme-linked immunosorbent assay. Nuclear translocation of the NF-κB p65 induced by LPS was observed by immunofluorescence staining.RESULTS:PANC-1 cancer cells showed preferential tumour cell ECM adhesion and ECM invasion compared with PANC-1 RNAi TLR4 cells, and this was enhanced by LPS. These effects were abolished by shTLR4 and NF-κB inhibition. LPS upregulates Integrinβ1 and MMP-9 in a dose-dependent and time-dependent manner, and the expression of Integrinβ1 and MMP-9 was decreased by NF-kB inhibition and shRNA. Nuclear translocation of the NF-κB p65 is induced by LPS in PANC-1.CONCLUSION:In pancreatic cancer, LPS promotes tumour cell ECM adhesion and invasion through activation of Integrinβ1 and MMP-9 in a TLR-4- and NF-κB-dependent manner.
|
PDF Full Text Request