慢性丙型肝炎病毒对肝癌细胞静止的影响及其机制研究

Effect of HCV on hepatocellular carcinoma cell quiescence and its underlying mechanism

摘要:

目的 探讨慢性丙型肝炎病毒（HCV）通过PCNA钳位相关因子（PCLAF）调控肝癌细胞静止的潜在机制。方法 HCV感染后，检测肝癌细胞HUH7的细胞周期分布。HCV感染或不感染后，肝癌细胞进行RNA-seq，使用siRNA敲低差异基因并检测细胞周期分布。过表达PCLAF后检测肝癌细胞的细胞周期分布。过表达HCV的非结构蛋白5A（NS5A）后，检测肝癌细胞PCLAF的表达和细胞周期分布。PCLAF抑制剂α-常春藤皂苷处理后，检测HCV对肝癌细胞周期分布的影响。结果 HCV感染后，肝癌细胞G0期和G1期细胞分布减少（P <0.05），而S期和G2/M期细胞分布增加（P <0.05）。敲低PCLAF后肝癌细胞G0期细胞分布增加（P <0.05）。HCV感染的情况下，过表达PCLAF后肝癌细胞G0期细胞分布减少（P <0.05）。过表达NS5A后，肝癌细胞PCLAF mRNA和蛋白相对表达量升高（P <0.05）。过表达NS5A后，肝癌细胞G0期和G1期细胞分布减少（P <0.05），而S期和G2/M期细胞分布增加（P <0.05）。但过表达NS5A的同时敲低PCLAF后，肝癌细胞的细胞周期分布无显著变化。过表达NS5A同时α-常春藤皂苷处理后，肝癌细胞的细胞周期无显著变化。HCV感染同时α-常春藤皂苷处理后，肝癌细胞的细胞周期无显著变化。结论 HCV诱导肝癌细胞跨越G0/G1期静止，减少细胞分裂时间。HCV的NS5A蛋白通过提高PCLAF的表达而抑制肝癌细胞静止。PCLAF抑制剂α-常春藤皂苷可以阻断HCV抑制的肝癌细胞静止。

Abstract:

Objective To explore the potential mechanism by which hepatitis C virus (HCV) regulates hepatocellular carcinoma (HCC) cell quiescence through PCNA clamp associated factor (PCLAF).Methods The cell cycle distribution of HCC cell line HUH7 was detected after HCV infection. RNA-seq was performed on HCC cells infected with HCV or not, and the cell cycle distribution was detected following knockdown of differential genes using siRNA or overexpression of PCLAF. After overexpression of HCV nonstructural protein 5A (NS5A), the expression of PCLAF in and the cell cycle distribution of HCC cells were determined. The effect of HCV on the cell cycle distribution of HCC cells was observed after treatment with the PCLAF inhibitor α-hederin.Results After HCV infection, the percentages of HCC cells in the G0 phase and the G1 phase were decreased (P < 0.05), while those in the S phase and the G2/M phase were increased (P < 0.05). The percentage of HCC cells in the G0 phase was increased after knockdown of PCLAF (P < 0.05). In the case of HCV infection, the percentage of HCC cells in the G0 phase was decreased after overexpression of PCLAF (P < 0.05). Following overexpression of NS5A, the relative mRNA and protein expressions of PCLAF in HCC cells were increased (P < 0.05), the percentages of HCC cells in the G0 phase and the G1 phase were decreased (P < 0.05), and the percentages of HCC cells in the S phase and the G2/M phase were increased (P < 0.05). However, the cell cycle distribution was not significantly changed after overexpression of NS5A and knockdown of PCLAF. Combined overexpression of NS5A and treatment with α-hederin and combined HCV infection and treatment with α-hederin also did not alter the cell cycle distribution of HCC cells.Conclusions HCV induces HCC cells to skip the quiescence in the G0/G1 phase and to reduce the time for cell division. HCV NS5A protein inhibits HCC cell quiescence by increasing the expression of PCLAF, which could be blocked by the PCLAF inhibitor α-hederin.