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目的研究线粒体融合素基因-2(mitofusin-2,Mfn2)沉默对人肝癌细胞株(HepG2)葡萄糖代谢和胰岛素信号通路分子丝/苏氨酸蛋白激酶(Akt1)的影响。方法构建Mfn2短发夹双链RNA(Mfn2shRNA)和阴性对照短发夹双链RNA(HK),将HepG2细胞株分为空白对照组(NC)、阴性对照组(HK)、Mfn2shRNA质粒转染组(Mfn2)。HK和Mfn2组细胞应用lipo-fectamine2000转染HepG2细胞株;采用葡萄糖氧化酶的方法和氚标记葡萄糖(3-3H-Glucose)放射性核素示踪法检测细胞葡萄糖消耗量和摄取率;Western blotting检测Mfn2和Akt1蛋白表达。结果与HK组比较,Mfn2组细胞Mfn2蛋白表达明显下降(0.23±0.05 vs 0.51±0.14,P=0.034),Akt1表达差异无统计学意义(0.13±0.00 vs 0.14±0.01,P=0.055);Mfn2组细胞葡萄糖消耗量明显下降(8.72±0.62 vs 9.75±0.35,P=0.001),葡萄糖摄取率亦显著下降(7.94±0.04 vs 9.64±0.13,P=0.002)。结论抑制Mfn2基因表达导致HepG2细胞葡萄糖代谢下降;Mfn2表达对葡萄糖代谢的影响是否通过PI3K/Akt信号通路介导还需要进一步研究。
Objective To investigate the effects of mitofusin-2 (Mfn2) silencing on glucose metabolism and insulin signaling pathway molecular filament / threonine protein kinase (Akt1) in human hepatocellular carcinoma cell line HepG2. Methods Mfn2 short hairpin double stranded RNA (Mfn2shRNA) and negative control short hairpin double stranded RNA (HK) were constructed. HepG2 cells were divided into blank control group (NC), negative control group (HK), Mfn2shRNA plasmid transfection group (Mfn2). The cells of HK and Mfn2 group were transfected into HepG2 cell line by lipo-fectamine 2000. The glucose consumption and uptake rate of cells were detected by glucose oxidase method and tritiated glucose (3-3H-Glucose) radionuclide. Western blotting Mfn2 and Akt1 protein expression. Results Compared with HK group, the expression of Mfn2 protein in Mfn2 group was significantly decreased (0.23 ± 0.05 vs 0.51 ± 0.14, P = 0.034), but there was no significant difference in Akt1 expression between the two groups (0.13 ± 0.00 vs 0.14 ± 0.01, P = 0.055) There was a significant decrease in glucose consumption (8.72 ± 0.62 vs 9.75 ± 0.35, P = 0.001) and glucose uptake (7.94 ± 0.04 vs 9.64 ± 0.13, P = 0.002). Conclusion Inhibition of Mfn2 gene expression leads to decreased glucose metabolism in HepG2 cells. Whether Mfn2 expression affects glucose metabolism through PI3K / Akt signaling pathway needs further study.