The regulation of hypoxia inducible factors on glucose transporter 1, pyruvate kinase M2 and vascular endothelial growth factor in BGC-823 cells

Abstract

Objective To compare the expression of glucose transporter 1 (GLUT1), pyruvate kinase M2 (PKM2), vascular endothelial growth factor (VEGF) in BGC-823 cells after transfected with small interfering RNA of hypoxia-inducible factors (HIF-1α, HIF-2α, HIF-1β) and investigate the effect of hypoxia inducible factors (HIFs) on the regulation of GLUT1, PKM2 and VEGF in gastric cancer. Methods Small RNA interference (RNAi) was used to silence BGC-823 cells. The mRNA and protein expression levels of HIFs, GLUT1, PKM2 and VEGF were detected by quantitative real-time reverse transcription polymerase chain reaction (PCR) and Western blotting. Results The mRNA (0.372±0.097) and protein (0.351±0.041) expression of HIF-1α was lower than in empty plasmid group (0.980±0.115 and 0.520±0.030) and blank control group (1.000±0.000 and 0.478±0.062) after infection with RNAi, the differences were all statistically significant (F=50.358, P=0.000; F=10.931, P=0.010). The mRNA (0.391±0.091) and protein (0.293±0.031) expression of HIF-2α was lower than in empty plasmid group (0.980±0.115 and 0.520±0.030) and blank control group (1.000±0.000 and 0.430±0.056) after infection with RNAi, the differences were all statistically significant (F=72.416, P=0.000; F=9.016, P=0.016). The mRNA (0.383±0.091) and protein (0.323±0.038) expression of HIF-1β was lower than in empty plasmid group (0.987±0.100 and 0.445±0.059) and blank control group (1.000±0.000 and 0.457±0.040) after infection with RNAi. The differences were all statistically significant (F=78.273, P=0.000; F=7.636, P=0.022). The mRNA (GLUT1: 0.721±0.067, 0.833±0.059, 0.493±0.067; VEGF: 0.711±0.057, 0.826±0.054, 0.793±0.068) and protein (GLUT1: 0.263±0.072, 0.303±0.042, 0.198±0.057; VEGF: 0.391±0.049, 0.451±0.054, 0.473±0.086) expression of GLUT1 and VEGF was all decreased after silencing HIFs (HIF-1α, HIF-1β, HIF-2α) when compared with empty plasmid group (mRNA: 1.046±0.094, 1.039±0.092; protein: 0.391±0.064, 0.690±0.066) and blank control group (mRNA: 1.000±0.000, 1.000±0.000; protein: 0.438±0.055, 0.707±0.040) and the differences were all statistically significant (VEGF mRNA: F=35.232, P=0.000; GLUT1 mRNA: F=15.364, P=0.000; VEGF protein: F=8.150, P=0.003, GLUT1 protein: F=17.109, P=0.000). The levels of VEGF decreased the most obvious when transfection with HIF-2α siRNA, while the levels of GLUT1 decreased the most obvious when transfection with HIF-1α siRNA. The mRNA (0.611±0.036) and protein (0.351±0.047) expression of PKM2 was all decreased after silencing HIF-1α when compared with empty plasmid group (1.037±0.197 and 0.731±0.057) and blank control group (1.000±0.000 and 0.737±0.043), the differences were all statistically significant, (F=12.480, P=0.007; F=60.403, P=0.000). Whlie only mRNA (0.793±0.067) decreased after silencing HIF-2α, the differences were statistically significant (F=12.480, P=0.007). The protein changed a little (0.675±0.030, F=1.211, P=0.373), and both mRNA (0.980±0.115) and protein (0.641±0.076) had no significant change after transfection with HIF-1β (F=0.144, P=0.869; F=2.404, P=0.171). Conclusion The results suggest that HIF-1α, HIF-2α and HIF-1β are all involved in regulating GLUT1 and VEGF. HIF-1α pathway plays a major role in regulating PKM2 and HIF-2α may regulate PKM2 in transcription level. Key words: Gastric cancer; Hypoxia-inducible factors; Pyruvate kinase M2; Glucose transporter 1; Vascular endothelial growth factor