HIF-3α对肝细胞癌多药耐药的影响及其机制

Effect of HIF-3α on multidrug resistance of hepatocellular carcinoma

目的探讨缺氧诱导因子3α(HIF-3α)对肝细胞癌多药耐药的影响及其机制。方法构建HIF-3α过表达慢病毒并鉴定;包装慢病毒,并测定慢病毒滴度。体外常规培养HepG2细胞,选择传2代、对数生长期、生长状态良好细胞,随机分为HIF-3α组、阴性对照组、空白对照组。HIF-3α组和阴性对照组分别转染HIF-3α过表达慢病毒、GV287空载体,空白对照组不予转染。收集各组转染72 h细胞,采用real-time PCR法检测HIF-3α、多药耐药基因1(MDR1)、多药耐药相关蛋白2(MRP2)、肺耐药蛋白(LRP)、谷胱甘肽硫转移酶pi(GST-pi)、拓扑异构酶Ⅱα(TopoⅡα)mRNA表达。结果本研究成功构建了HIF-3α过表达慢病毒,其测序结果与GeneBank中HIF-3α的基因编码序列完全一致。该基因编码序列慢病毒包装后,荧光显微镜下可见大量绿色荧光,说明慢病毒包装成功,经计算,慢病毒滴度为1.0×10~8 TU/mL。HIF-3α组HIF-3αmRNA相对表达量明显高于阴性对照组和空白对照组(P均<0.05),而阴性对照组与空白对照组比较P>0.05。HIF-3α组GST-pi、LRP mRNA相对表达量均明显高于空白对照组和阴性对照组(P均<0.05),MDR1、MRP2、TopoⅡαmRNA相对表达量组间两两比较P均>0.05。结论 HIF-3α可能与肝细胞癌多药耐药有关,其机制与上调多药耐药相关基因GST-pi、LRP表达有关。

Objective To investigate the effect of hypoxia-inducible factor 3α(HIF-3α)on multidrug resistance of hepatocellular carcinoma and its mechanism.Methods Lentiviral vectors overexpressing HIF-3αwere constructed and identified,and the lentiviruses were packaged and the lentiviral titers were determined.HepG2 cells were routinely cultured in vitro.The cells of the second generation,in logarithmic growth phase and with good growth status were randomly divided into the HIF-3αgroup,negative control group,and blank control group.The cells in the HIF-3αgroup and negative control group were transfected with HIF-3αoverexpression lentivirus and GV287 empty vector,respectively,and the cells in the blank control group were not transfected.The cells were collected at 72 h after transfection,and we detected the HIF-3α,multidrug resistance gene 1(MDR1),multidrug resistance-associated protein 2(MRP2),lung resistance protein(LRP),and glutathione S-transferase Pi(GST-pi),and topoisomeraseⅡα(TopoⅡα)mRNA expression by real-time PCR.Results The HIF-3αoverexpression lentivirus was successfully constructed and its sequencing results were consistent with the gene coding sequence of HIF-3αin GeneBank.After the coding sequence of the gene was packaged by lentivirus,a large amount of green fluorescence was observed under the fluorescence microscope,indicating that the lentiviral packaging was successful,and the lentivirus titer was calculated to be 1.0×10~8 TU/mL.The relative expression of HIF-3αmRNA in the HIF-3αgroup was significantly higher than that in the negative control group and blank control group(both P<0.05),while no significant difference was found between the negative control group and the blank control group(P>0.05).The relative expression levels of GST-pi and LRP mRNA in the HIF-3αgroup were significantly higher than those in the blank control group and negative control group(all P<0.05).There was no significant difference in the MDR1,MRP2,and TopoⅡαmRNA expression between every two groups(all P>0.05).Conclusion HIF-3αmay be associated with multidrug resistance in hepatocellular carcinoma,and its mechanism is related to the up-regulation of GST-pi and LRP expression.