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首页> 外文期刊>World Journal of Gastroenterology >Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line
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Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line

机译：细胞外信号调节激酶/有丝分裂原激活的蛋白激酶途径与HBx诱导的肝癌细胞多药耐药性的关系

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AIM: To investigate the molecular mechanism of the influence of HBx protein on multidrug resistance associated genes: multidrug resistance 1 (MDR-1), multidrug related protein (MRP-1), lung resistance related protein (LRP) in hepatoma cells and the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process. METHODS: A cell model stably expressing the HBx protein was established by liposome-mediated transfection of HBx gene into HepG2 cell line. The expression of multidrug resistance associated genes and proteins was detected by RT-PCR and Western blot. AnnexinV-FITC/PI assay was used to confirm the multidrug resistance (MDR) phenotype of transfected cells by fluorescence cytometry (FACS). The ERK/MAPK pathway activation was measured by Western blot through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein. After treated with the ERK/MAPK pathway inhibitor U0126, the HBx-expressing cells were harvested. Then RT-PCR, Western blot and FACS were used to analyze the alterations in the expression of multidrug resistance associated genes and the MDR phenotype after exposure. RESULTS: Compared with the control group, the transfected cells showed a higher expression of MDR associated genes and proteins. Marked elevations in MDR-1 (64.3%), MRP-1 (87.5%) and LRP (90.8%) were observed in the transfected cells (P < 0.05). RT-PCR revealed that the over-expression of MDR associated proteins was due to amplification of such genes (MDR1 2.9 fold, MRP1 1.67 fold, LRP1.95 fold). Furthermore, we found that the ERK/MAPK activity was remarkably high in the HBx-expressing cells. The activation of ERK/MAPK, as measured by the ratio of phosphorylated ERK bands normalized to the total ERK bands, was increased by 2.3-fold in HBx-transfected cells compared with cells transfected with the empty vector. After treated with the ERK/MAPK pathway inhibitor, the level of MDR associated genes and proteins in the transfected cells decreased to some extent. Compared with controls, a significant decrease in MDR-1 mRNA (53.3%), MRP-1 mRNA (59.7%) as well as LRP mRNA (56.4%) was observed in the U0126 treated transfected cells after 12 h. Western blot also demonstrated that the protein expression of these MDR associated genes slightly reduced after treated with U0126 for 12 h (MDR-1 40.1%, MRP-1 29.4%, LRP35.7%). This change was accompanied with the rise of cell apoptosis ratio confirmed by Annexin V-PI detection. The apoptosis index of U0126-treated cells increased by 1.28 fold, compared with that of transfected cells. Obviously, the MDR phenotype of these cells was obviously related with increased activities of the ERK/MAPK pathway. CONCLUSION: HBx protein might be one of the causes for the occurrence of MDR in HCC, and ERK/MAPK pathway might be involved in this change.
机译：目的：探讨HBx蛋白影响多药耐药相关基因的分子机制：多药耐药1（MDR-1），多药相关蛋白（MRP-1），肝癌细胞中肺耐药相关蛋白（LRP）的潜力细胞外信号调节激酶/促分裂原活化蛋白激酶（ERK / MAPK）通路在此过程中的作用。方法：通过脂质体介导的HBx基因转染HepG2细胞系，建立稳定表达HBx蛋白的细胞模型。通过RT-PCR和Western blot检测多药耐药相关基因和蛋白的表达。使用AnnexinV-FITC / PI测定法通过荧光细胞术（FACS）确认转染细胞的多药耐药性（MDR）表型。通过比较ERK / MAPK的磷酸化与总ERK / MAPK蛋白的比率，通过蛋白质印迹法测量ERK / MAPK途径的活化。用ERK / MAPK途径抑制剂U0126处理后，收获表达HBx的细胞。然后采用RT-PCR，Western blot和FACS分析多重耐药相关基因的表达和暴露后MDR表型的变化。结果：与对照组相比，转染细胞的MDR相关基因和蛋白表达更高。在转染的细胞中观察到MDR-1（64.3％），MRP-1（87.5％）和LRP（90.8％）明显升高（P <0.05）。 RT-PCR显示MDR相关蛋白的过表达是由于此类基因的扩增（MDR1 2.9倍，MRP1 1.67倍，LRP1.95倍）。此外，我们发现在HBx表达细胞中ERK / MAPK活性非常高。与用空载体转染的细胞相比，用HBx转染的细胞将ERK / MAPK的活化（标准化为总ERK带的磷酸化ERK带的比率）提高了2.3倍。用ERK / MAPK途径抑制剂处理后，转染细胞中MDR相关基因和蛋白质的水平有所降低。与对照组相比，在用U0126处理的转染细胞12小时后，观察到MDR-1 mRNA（53.3％），MRP-1 mRNA（59.7％）和LRP mRNA（56.4％）的显着降低。蛋白质印迹法还表明，在用U0126处理12小时后，这些MDR相关基因的蛋白质表达略有降低（MDR-1 40.1％，MRP-1 29.4％，LRP35.7％）。这种变化伴随着膜联蛋白V-PI检测所证实的细胞凋亡率上升。与转染的细胞相比，U0126-处理的细胞的凋亡指数增加了1.28倍。显然，这些细胞的MDR表型明显与ERK / MAPK途径的活性增加有关。结论：HBx蛋白可能是肝癌发生MDR的原因之一，其变化可能与ERK / MAPK途径有关。

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