Mechanism of lipid rafts in Toll-like receptor 4 in promoting the high activity of hypoxia-inducible factor-1α in cervical cancer

Abstract

Objective To investigate the role and mechanism of lipid rafts in Toll like receptor(TLR) 4 in promoting the high activity of hypoxia inducible factor(HIF)-1α in cervical cancer. Methods In vitro cell experiments have been conducted. Cervical cancer Siha cells were randomly divided into 4 groups according to the computer randomized method, and then treated with the following methods, methyl beta cyclodextrin(MβCD)+ lipopolysaccharide group was treated with MβCD and lipopolysaccharide stimulation, siTLR4+ lipopolysaccharide group was treated with siTLR4 transfection and lipopolysaccharide stimulation.The blank control group was not treated and stimulated, and lipopolysaccharide control group only treated with lipopolysaccharide stimulation.Detected and observed the following items in 4 groups: ①MTT colorimetric method was used to measure the optical density(OD) value of cervical cancer Siha cells suspension and drawn the Siha cells growth inhibition curves. Colony formation was observed in soft agar colony culture. ②The intracellular positive expression and content variations of HIF-1α protein of cervical cancer Siha cells were detected by immunocytochemical staining and Western blotting. ③Lucigenin nicotinamide adenine dinucleotide phosphate(NADPH) oxidase activity in cervical cancer Siha cells was detected with chemiluminescence and intracellular reactive oxygen species with DCFH-DA probe. ④The colocalization of lipid rafts and HIF-1α protein was detected by immunofluorescence staining.The differences of these items above mentioned among groups and at different time points(0, 12, 24, 36, 48 h) after threated and stimulation were compared by statistical method. Results ① The MTT assay and soft agar experiments showed that, the activity of cervical cancer Siha cells and colony numbers of lipopolysaccharide control group were higher or more than those of blank control group, while those of MβCD + lipopolysaccharide group and siTLR4 + lipopolysaccharide group were lower or less than those of lipopolysaccharide control group or blank control group, and all the differences were statistically significant (P<0.01). ② The immunocytochemical staining and Western blotting showed that, the relative expression of HIF-1α protein of cervical cancer Siha cells in lipopolysaccharide control group was more than that of blank control group, while that of MβCD + lipopolysaccharide group and siTLR4 + lipopolysaccharide group were less than that of blank control group or lipopolysaccharide control group, and all the differences were statistically significant (P<0.05). ③ NADPH oxidase activity and reactive oxygen content in cervical cancer Siha cells in MβCD+ lipopolysaccharide group and siTLR4 + lipopolysaccharide group after treated at 12, 24, 36, 48 h were lower than those of blank control group or lipopolysaccharide control group at the same time point. The activity of NADPH oxidase and reactive oxygen content in cervical cancer Siha cells in lipopolysaccharide control group after treated at 24, 36, 48 h were higher than those of blank control group at the same time point, and the differences were statistically significant(P<0.05). ④ The immunofluorescence staining showed that, lipid rafts number located on the cell surface with green fluorescence, and HIF-1α protein was mainly located in the cytoplasm with red fluorescence. The cells with the increased lipid rafts number were also showed the high expression level of HIF-1α protein. Conclusions TLR4 signal may activate the lipid raft and stimulate the redox signal of NADPH oxidase, and then produce reactive oxygen to maintain the high activity of HIF-1α in cervical cancer cells. Key words: Uterine cervical neoplasms; Membrane microdomains; Toll-like receptor 4; Reactive oxygen species