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

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ATG5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q RT-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ATG5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G_1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G_1期,可能与对抗缺血引起的死亡有关。

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 Hepa1-6was 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（q RT-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.