ATG5 介导肝癌细胞对抗营养危机的作用研究

Effect of ATG5-mediated anti-nutritional crisis in hepatocellular carcinoma

摘要:

目的 探索在小鼠肿瘤细胞中对抗营养缺乏的分子途径。方法 构建ATG 5 基因表达的质粒并瞬 时转染至细胞内；用不同靶基因的si-RNAs 转染到细胞来沉默基因的表达；不同条件下提取小鼠正常肝细胞 AML-12 和肝癌细胞Hepa1-6 的RNA 和蛋白；用qRT-PCR 检测基因的表达；用Western blot 检测蛋白的表达； 用流式细胞仪检测细胞周期；用CCK-8 的试剂测定细胞活性。结果 与小鼠正常肝细胞AML-12 比较，肝癌 细胞Hepa1-6 对缺血引发的细胞死亡有抵抗性。缺血使Hepa1-6 细胞内诱导自噬相关蛋白ATG5 表达升高 （P <0.05）；沉默内源ATG 5 基因后Hepa1-6 细胞对缺血的敏感性升高，且ATG5 诱导细胞周期抑制蛋白p21（cip1/ waf1）表达增加使细胞滞留在G1 期。结论 在营养缺乏的环境中，Hepa1-6 细胞内ATG5 表达升高，且ATG5 增加p21（cip1/waf1）的表达量使细胞滞留在G1 期，可能与对抗缺血引起的死亡有关。

Abstract:

Objective To investigate the effect of anti-nutritional crisis in tumor cells and potential molecular mechanisms. Methods Normal mouse liver cell AML-12 as well as hepatocellular carcinoma cell line Hepa1-6 was utilized in this study. ATG5 expressing construct was transfected into cells. Si-RNA technology was performed for silencing of targeted genes. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis and Western blot were carried for RNA levels and proteins, respectively. Cell cycle status and cellular activity were identified by flow cytometry and CCK-8 kit, respectively. Results Hepa1-6 was more resistant to nutrition deprivation compared with AML-12. Serum withdrawal induced remarkable increased expression of ATG5 in Hepa1-6 cells (P < 0.05). Knockdown of ATG5 reversed resistance of cells to nutrition deprivation. Further, ATG5 induced increase of p21 (cip1/waf1), which rested the Hepa1-6 cells in G1 phase. Conclusion Resistance of tumor cells to nutrient deficiency may be achieved through ATG5-p21 (cip1/waf1) signaling pathway which rests cells on G1 stage.