胃癌BGC-823细胞中缺氧诱导因子对葡萄糖转运蛋白1、M2型丙酮酸激酶及血管内皮生长因子的调控作用

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

目的观察小干扰RNA（siRNA）特异性沉默人胃癌BGC-823细胞中缺氧诱导因子（HIF-1α、HIF-2α、HIF-1β）后葡萄糖转运蛋白1（GLUT1）、M2型丙酮酸激酶（PKM2）及血管内皮生长因子（VEGF）的表达，探讨HIFs对胃癌细胞内GLUT1、PKM2及VEGF的调控差异。 方法用实时定量反转录聚合酶链反应（RT-qPCR）及Western blot法检测转染HIFs及Control小干扰RNA（siRNA）后细胞内GLUT1、PKM2及VEGF的表达。 结果RNA干扰技术沉默HIF-1α后其mRNA、蛋白表达量分别为0.372±0.097、0.351±0.041，均低于空载体组（0.980±0.115、0.520±0.030）及空白对照组（1.000±0.000、0.478±0.062），差异均有统计学意义（F＝50.358，P＝0.000；F＝10.931，P＝0.010）；RNA干扰技术沉默HIF-2α后其mRNA、蛋白表达量分别为0.391±0.091、0.293±0.031，均低于空载体组（1.037±0.090、0.427±0.026）及空白对照组（1.000±0.000、0.430±0.056），差异均有统计学意义（F＝72.416，P＝0.000；F＝9.016，P＝0.016）；RNA干扰技术沉默HIF-1β后其mRNA、蛋白表达量分别为0.383±0.091、0.323±0.038，均低于空载体组（0.987±0.100、0.445±0.059）及空白对照组（1.000±0.000、0.457±0.040），差异均有统计学意义（F＝78.273，P＝0.000；F＝7.636，P＝0.022）。转染HIFs（HIF-1α、HIF-1β、HIF-2α） siRNA后VEGF及GLUT1在mRNA（分别为0.721±0.067、0.833±0.059、0.493±0.067；0.711±0.057、0.826±0.054、0.793±0.068）及蛋白（分别为0.263±0.072、0.303±0.042、0.198±0.057；0.391±0.049、0.451±0.054、0.473±0.086），表达水平较空载组（mRNA：1.046±0.094、1.039±0.092；蛋白：0.391±0.064、0.690±0.066）及对照组（mRNA：1.000±0.000、1.000±0.000；蛋白：0.438±0.055、0.707±0.040）下降，差异均有统计学意义（VEGF mRNA：F＝35.232，P＝0.000；GLUT1 mRNA：F＝15.364，P＝0.000；VEGF蛋白：F＝8.150，P＝0.003，GLUT1蛋白：F＝17.109，P＝0.000），其中转染HIF-2α后VEGF表达下调最明显，转染HIF-1α后GLUT1表达下调最明显。转染HIF-1α后PKM2在mRNA（0.611±0.036）及蛋白（0.351±0.047）表达水平较空载体组（1.037±0.197、0.731±0.057）及对照组（1.000±0.000、0.737±0.043）均下降，差异均有统计学意义（F＝12.480，P＝0.007；F＝60.403，P＝0.000）。转染HIF-2α后PKM2 mRNA（0.793±0.067）表达下降，差异均有统计学意义（F＝12.480，P＝0.007），蛋白无明显变化（0.675±0.030，F＝1.211，P＝0.373）；而转染HIF-1β（mRNA：0.980±0.115，蛋白：0.641±0.076）后PKM2无明显下降（F＝0.144，P＝0.869；F＝2.404，P＝0.171）。 结论研究结果提示HIF-1α、HIF-2α和HIF-1β均参与调控GLUT1、及VEGF的表达，HIF-1α参与调控PKM2，HIF-2α在转录水平可能调控PKM2的表达。

ObjectiveTo 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. MethodsSmall 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. ResultsThe 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）. ConclusionThe 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.