The Effcet Of HIF-1α In The Regulation Of TLR4in Pancreatic Cancer Cells In Vitro And In Vivo

Objective: To investigate the relationship of Toll-like receptor4(TLR4) and hypoxiainducible factor-1α (HIF-1α) expression in pancreatic cancer.Methods: We used immunohistochemical staining to examine the expression of TLR4and HIF-1α in84cases of pancreatic cancer. The relationship between TLR4andpathologic features, association between TLR4and HIF-1α were also analyzed. Cellswere placed air-tight chamber or treated with CoCl2to mimic tumor hypoxic microenvironment. Real-time PCR and immunofluorescence staining or flow cytometryanalysis were preformed to investigate the effects of hypoxia on the expression ofTLR4in pancreatic cancer cells.Results: Immunohistochemical staining showed that the positive rates of TLR4andHIF-1α expression were76%and79%in84cases of pancreatic cancer. Regionaldistribution of TLR4protein mostly locatedly overlapped with HIF-1α in consecutivesections of same pancreatic cancer tissue. Spearman analysis indicated that TLR4expression strongly correlated with HIF-1α in pancreatic cancer (r=0.623, P<0.001).Both hypoxia and CoCl2enhanced the levels of TLR4mRNA in a time-dependentmanner. Protein levels of TLR4were markedly increased after exposure to hypoxia orCoCl2for12h(P<0.05).Conclusion: Both of TLR4and HIF-1α were expressed with a different degree inpancreatic cancer tissue and cells, and their expression had a strong relationship. Objective: To observe the effect of HIF-1α in the regulation of TLR4expression inpancreatic cancer cells under hypoxic conditions.Methods: In this study, we intended to construct HIF-1α siRNA plasmid and HIF-1αexpressing plasmid and then transfected into Panc-1cells. Cells were placed air-tightchamber or treated with CoCl2to mimic tumor hypoxic micro environment. ThemRNA and protein levels of HIF-1α in the stable-transfected cells were detected byRT-PCR and Western blot assays. Protein and mRNA levels of TLR4were detectedby flow cytometric analysis and RT-PCR.Results: We successfully constructed HIF-1α siRNA plasmid and HIF-1α expressingplasmid plasmid, and the mRNA and protein levels of HIF-1α in the HIF-1αoverexpression cells were significantly increased while the levels were decreased inHIF-1α knockdown cells in cells under hypoxic conditions. Over-expression ofHIF-1α increased expression of the luciferase reporter that was driven by the TLR4promoter and the TLR4mRNA levels. Up-regulation of TLR4mRNA and proteinexpression induced by hypoxia or CoCl2, was significantly attenuated when HIF-1αexpression was knocked down by siRNA.Conclusion: These results suggest that TLR4expression in pancreatic cancer cells isup-regulated via HIF-1α in response to hypoxic stress. Objective: To explore the inhibitory effects of pSil2.1_hygro-1589shRNA targetingHIF-1α on the growth of Human Pancreatic Carcinoma Xenograft Tumors in nudeMice and its influences on expression of TLR4.Methods: In this study, the stably transfected Panc-1cell line and control cells wereinjected s.c. in athymic nude mice. Each cells were injected subcutaneously into theflank of8-week-old male BALB/c nude mice. For each group,12animals were used.Tumors were allowed to establish for2weeks and then their size was measured withcalipers twice weekly for up to8weeks. After8weeks, all mice were sacrificed andtake the intact subcutaneous tumor tissues, and then measure their volumes and theirweight. Tumor growth inhibitory rate was calculated finally. Immunohistochemistryassay was performed to detect the expression levels of TLR4protein．Results: We found that there was statistical difference in tumor growth between thecontrol group and the HIF-1α shRNA group after30days (P<0.05). In HIF-1αshRNA group, the subcutaneous tumors grew slowly, the meanvolume(125.06±14.12mm3)and mean weight(0.189±0.025g)were significantlydifferent from the control group(2125.06±89.56mm3,2.89±0.75g)(P<0.01)and thetumor growth inhibitory rate was94.2％in HIF-1α shRNA group. The expressionlevels of TLR4protein in tumor tissues of HIF-1α shRNA group were obviouslylower compared with control-shRNA group and control group(P<0.05)Conclusion: Stable knockdown of HIF-1α significantly reduces tumor volume andthe expression of TLR4in vivo...