Synergistic Effect of MC-LR and C-Terminal Truncated HBx on HepG2 Cells and Their Effects on PP2A Mediated Downstream Target of MAPK Signaling Pathway.
Synergistic Effect of MC-LR and C-Terminal Truncated HBx on HepG2 Cells and Their Effects on PP2A Mediated Downstream Target of MAPK Signaling Pathway.
复制标题
MC-LR和C端截短的HBx对HepG2细胞的协同作用及其对PP2A介导的MAPK信号通路下游靶点的影响
DOI：
10.3389/fgene.2020.537785
复制
发表时间：
2020
影响因子：
3.7
通讯作者：
Deng W
中科院分区：
文献类型：
--
作者：
Xiao C;Mei F;Ren G;Long L;Chen M;Fang X;Li J;Li K;Tang Y;Huang T;Deng W
C端截短的乙型肝炎病毒（HBV）X（ctHBx）感染和接触微囊藻毒素-LR（MC-LR）可导致人类肝炎和肝癌，但其协同作用的机制尚未完全阐明。构建ctHBx（HBxΔ4和HBxΔ32）慢病毒并转染到HepG2细胞中。然后采用酶联免疫吸附实验、克隆形成实验、划痕实验、Transwell实验等分子生物学方法研究MC-LR和ctHBx的功能，分别进行流式细胞术检测各组细胞周期和凋亡，并通过丝裂原激活蛋白激酶（MAPK）途径检测HBx、MEK/ERK/JNK/p38以及下游蛋白cdc2、cdc25C、p53等。蛋白质印迹法。我们发现，在同一时间点和MC-LR浓度下，MC-LR和HBxΔ32/HBxΔ4组的蛋白磷酸化酶2A（PP2A）酶活性比MC-LR和HBx组下降幅度更大（P < 0.05）。同时MC-LR组和ctHBx组HepG2细胞的增殖、迁移、侵袭和集落形成能力均显着增强（P < 0.05）。此外，MC-LR处理的HBxΔ32/HBxΔ4组中S期细胞的比例显着高于未处理组（P < 0.05）。此外，MC-LR和HBxΔ32上调MAPK信号通路包括磷酸化MEK1/2、ERK1/2、p38和JNK的蛋白表达，并且细胞周期蛋白相关蛋白包括p53、cdc25C和cdc2的表达也被激活(P < 0.05)。综上所述，我们的研究结果揭示了 PP2A/MAPK/p53、cdc25C 和 cdc2 轴上的 MC-LR 和 ctHBx 在 HCC 形成和发展中的重要意义，并确定 MC-LR 和 ctHBx 是肝癌发生的潜在因果辅助因子。
C-terminally truncated hepatitis B virus (HBV) X (ctHBx) infection and exposure to microcystins-LR (MC-LR) can lead to human hepatitis and liver cancer, but the mechanism associated with their synergistically effects not been fully elucidated. The ctHBx (HBxΔ4 and HBxΔ32) lentivirus were constructed and transfected into the HepG2 cells. Then we investigated the function of MC-LR and ctHBx using the molecular biology approaches, including enzyme-linked immunosorbent assay, clone formation assay, scratch wound testing, transwell assays, carried out flow cytometry respectively to examine cell cycle and apoptosis in each group, and detected the related proteins of HBx, MEK/ERK/JNK/p38 in mitogen-activated protein kinase (MAPK) pathway and the downstream proteins such as cdc2, cdc25C, and p53 by western blotting. We found that the protein phosphorylase 2A (PP2A) enzyme activity in MC-LR and HBxΔ32/HBxΔ4 groups decreased more than in MC-LR and HBx group at the same time point and MC-LR concentration (P < 0.05). Meanwhile the proliferation, migration, invasion and colony formation capability of HepG2 cells were significantly enhanced in MC-LR and ctHBx groups (P < 0.05). In addition the proportion of S stage cells in the MC-LR-treated HBxΔ32/HBxΔ4 groups was significantly greater than that in the untreated groups (P < 0.05). Furthermore, the protein expression of MAPK signaling pathway including phospho-MEK1/2, ERKl/2, p38, and JNK were up-regulated by MC-LR and HBxΔ32, and the expression of cyclin-related proteins, including p53, cdc25C, and cdc2 were also activated (P < 0.05). Taken together, our findings revealed the essential significance of the MC-LR and ctHBx on the PP2A/MAPK/p53, cdc25C and cdc2 axis in the formation and development of HCC and identified MC-LR and ctHBx as potential causal cofactors of hepatocarcinogenesis.