HBP21抑制胰岛素诱导的PI3K/AKT信号通路机制

朱海珍 王鑫涛 许艳 田仁云 邓日林 王静静 陈生稳 李慧逸

摘   要：磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）信号通路在肝细胞癌中处于异常激活的状态，并且促进癌症的发生发展. 在肝细胞癌中，热休克蛋白HSP70結合蛋白21（Hsp70 binding protein 21，HBP21）处于低表达状态，过表达HBP21显著诱发癌细胞发生凋亡. 利用qRT-PCR技术检测肝癌组织中HBP21的mRNA水平，发现癌组织中HBP21的mRNA水平要低于癌旁组织. 用胰岛素处理肝癌细胞Huh7以激活细胞内PI3K/AKT信号通路，采用qRT-PCR技术和蛋白免疫印迹实验检测细胞内HBP21的转录和翻译水平，随着胰岛素处理时间的延长，HBP21的mRNA水平和蛋白水平都呈现下降趋势. 在Huh7内过表达HBP21显著抑制胰岛素诱导的AKT和哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）的磷酸化;通过泛素化实验和蛋白免疫印迹实验发现，HBP21不影响AKT的K48及K63位泛素化及人第10号染色体缺失的磷酸酶及张力蛋白同源的基因（phosphatase and tensin homolog deleted on chromosome ten，PTEN）的蛋白水平. 利用抑制剂LY294002处理Huh7细胞，确定HBP21作用于PI3K/AKT信号通路. 进一步研究发现，HBP21不影响IRS1和IRS2的mRNA水平，而是抑制胰岛素受体β亚基（insulin receptor beta，IRβ）的磷酸化进而影响PI3K/AKT信号通路的活化. 在Huh7细胞中，HBP21抑制肝癌细胞中异常活化的PI3K/AKT发挥着重要作用. HBP21在肝细胞癌中的缺失表达，以及其抑制PI3K/AKT信号通路使其有可能成为潜在治疗癌症的靶点.

关键词： HBP21;肝细胞癌;PI3K/AKT/mTOR信号通路;PTEN;酶

中图分类号：Q71                                文献标志码：A

HBP21 Inhibiting Insulin-induced PI3K/AKT Signal Pathway Mechanism

ZHU Haizhen1，2，3?，WANG Xintao1，2，3，XU Yan1，2，3，TIAN Renyun1，2，3，

DENG Rilin1，2，3，WANG Jingjing1，2，3，CHEN Shengwen1，2，3，LI Huiyi1，2，3

（1. College of Biology，Hunan University，Changsha 410082，China;

2. Institute of Pathogen Biology and Immunology，Hunan University，Changsha 410082，China;

3. State Key Laboratory of Chemo/Biosensing and Chemometrics，Hunan University，Changsha 410082，China）

Abstract：The phosphatidylinositol 3-kinase/protein kinase B （PI3K/AKT） signaling pathway is abnormally activated in hepatocellular carcinoma and promotes the occurrence and development of cancer. In hepatocellular carcinoma， the heat shock protein Hsp70 binding protein 21（Hsp70 binding protein 21） is in a low expression state， and overexpression of HBP21 significantly induces cancer cell apoptosis. Using qRT-PCR technology to detect the mRNA level of HBP21 in liver cancer tissues， it is found that the mRNA level of HBP21 in cancer tissues is lower than that in adjacent tissues. Hepatocellular carcinoma cell Huh7 is treated with insulin to activate the PI3K/AKT signaling pathway in the cells， and the transcription and translation levels of HBP21 in the cells are detected by qRT-PCR technology and Western blotting. With the prolonged insulin treatment time， the mRNA and protein levels of HBP21 show a downward trend. Overexpression of HBP21 in Huh7 cells significantly inhibits insulin-induced phosphorylation of AKT and mammalian target of rapamycin（mTOR）;through ubiquitination and western blotting experiments， it is found that HBP21 does not affect AKT K48 and K63 ubiquitination as well as phosphatase and tensin homolog deleted on chromosome ten（PTEN） protein levels. The inhibitor LY294002 was used to treat Huh7 cells to confirm that HBP21 acts on the PI3K/AKT signaling pathway. Further research shows that HBP21 does not affect the mRNA levels of IRS1 and IRS2， but inhibits the phosphorylation of insulin receptor beta（IRβ） and affects the activation of the PI3K/AKT signaling pathway. In Huh7 cells，HBP21 plays an important role in inhibiting abnormal activation of PI3K/AKT in liver cancer cells. The lack of expression of HBP21 in hepatocellular carcinoma and its inhibition of PI3K/AKT signaling pathway make it possible to become a potential target for cancer treatment.

Key words：Hsp70 binding protein 21（HBP21）;hepatocellular carcinoma（HCC）;PI3K/AKT/mTOR signaling pathway;phosphatase and tensin homolog deleted on chromosome ten（PTEN）;enzymes

肝细胞癌（hepatocellular carcinoma，HCC）是全球第六大常见恶性肿瘤，位居全球癌症相关死亡的第四位，一般认为肝癌的发生与慢性乙型和丙型肝炎病毒感染、酗酒或饮食中接触黄曲霉毒素有关[1-2]. 像许……