lncRNAHIPK1-AS1作为介导JNK/p38及HIPK1回路的关键分子在蜂毒素促肝癌细胞凋亡中的机制研究

Hepatocellular carcinoma (HCC) is not sensitive to many common treatments. A series of studies have proved that melittin has a therapeutic effect on hepatoma through multi-targets, multi-ways and multi-effects. On the basis of previous studies, this project intends to find the key molecules of HCC cell apoptosis and the markers of drug susceptibility can improve the application value of melittin. Our previous studies have shown HCC cells treated with melittin can down-regulate the expression of lncRNA HIPK1-AS1. Combining the preliminary experiment results with bioinformatics analysis, we speculate that melittin may up-regulate the expression of transcription factor ATF-3 by activating the JNK/p38 pathway, thus increasing ATF-3 binding to lncRNA HIPK1-AS1 at its promoter region and activating its expression. LncRNA HIPK1-AS1 can inhibit the activity of DNMT1 by binding with DNMT1 protein, hander the formation of DNA methylation at HIPK1 promoter region, up-regulate the expression of HIPK1 and activate its downstream signaling pathways—ASK1-JNK/p38, JNK/p38 lightning loop formation, promote cell apoptosis. It is promising to provide fundamental basis for the research about lncRNA HIPK1-AS1 as melittin sensitive marker.

肝癌对许多常规治疗手段不敏感。本课题组对蜂毒素抗肝癌效用进行的系列研究证实其可多靶点、多途径、多效应作用于肝癌细胞。本项目拟在既往研究的基础上，寻找蜂毒素的药物敏感标志物，进一步提高其应用价值。本项目前期研究表明，蜂毒素可下调肝癌细胞lncRNA HIPK1-AS1表达，结合预实验和生物信息学分析，我们推测其可能通过活化JNK/p38通路上调转录因子ATF-3的表达，ATF-3结合到lncRNA HIPK1-AS1启动子区激活该lncRNA的表达。而lncRNA HIPK1-AS1通过结合到DNMT1蛋白的活性位点抑制其酶的活性，阻碍HIPK1基因启动子区DNA甲基化形成，使HIPK1基因的表达上调，从而激活该基因的下游信号通路：ASK1-JNK/p38，形成JNK/p38回路，促进细胞凋亡。此研究，有望为lncRNA HIPK1-AS1作为蜂毒素的药物敏感标志物的研发提供理论基础。

许多长非编码RNA（lncRNAs）是肝细胞癌（HCC）发生发展的的关键调节因子，但大多数lncRNAs在HCC中的作用机制仍不明确。本课题着眼于，lncRNA ADAMTS9-AS1在肝癌中的效用。我们前期的qPCR分析结果中显示，肝癌细胞系中ADAMTS9-AS1的表达高于正常细胞。此外，CCK-8、划痕愈合、跨孔迁移和侵袭实验表明ADAMTS9-AS1过表达可显著促进MHCC97-H和HepG2细胞的增殖、迁移和侵袭；而将ADAMTS9-AS1敲除后，结果恰恰相反。根据GEO的检索发现，ADAMTS9-AS1与PI3K/AKT/mTOR通路相关。因此本课题，进一步探讨了ADAMTS9-AS1与PI3K/AKT/mTOR信号通路之间的关系。我们观察了该通路中p-AKT、PIK3CB和p-mTOR的蛋白表达水平的变化，Western-blot结果表明ADAMTS9-AS1可增强了该三种蛋白的表达水平；而他们作为PI3K/AKT/mTOR通路的关键调节分子，启发了我们进一步研究该通路与凋亡和自噬相关蛋白的关系。研究结果显示，ADAMTS9-AS1表达与LC3-II、BECN1和促凋亡的Bax呈负相关，而与SQSTM1和抗凋亡的Bcl-2表达呈正相关。而，Westernblot结果表明ADAMTS9-AS1可降低ADAMTS9的表达。因此总结此次研究结果，ADAMTS9-AS1参与了肝癌细胞的增殖、迁移和侵袭，其机制可能是由于激活了PI3K/AKT/mTOR信号通路，从而影响肝癌细胞的自噬和凋亡。这提示ADAMTS9-AS1也许在将来，会成为肝癌治疗的分子靶点。