摘要
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.
作者
曲颜丽
王海峰
唐勇
Qu Yanli;Wang Haifeng;Tang Yong(Department of Digestive Tumor, the Affiliated Tumor Hospital of Xinfiang Medical University, Urumqi 830011, China;Department of Radiotherapy of Chest and Abdomen, the Affilialed Tumor Hospital of Xinjiang Medical University, Urumqi 830011, Chin)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2018年第4期627-630,共4页
Chinese Journal of Experimental Surgery
基金
新疆维吾尔自治区自然科学基金(2015211C124)