长链非编码RNA AK096649在肝癌转移复发中的作用及分子机制研究

Hepatocellular carcinoma (HCC) has high incidence, high metastasis and recurrence rate, and short survival time, but the underlying molecular mechanism remains largely unknown. So it is of great significance to investigate the genes related to HCC and their molecular mechanism in metastasis and recurrence. Studies have shown that long noncoding RNA (lncRNA) is closely related to tumor metastasis and recurrence. In our previous studies, the expression of lncRNA AK096649 was found to up-regulate in 76.1% HCC tissues. The high expression of lncRNA AK096649 was correlated with the HCC metastasis, recurrence, poor survival, etc. Silencing AK096649 expression by RNA interference technology could significantly reduce the invasion and migration ability of HCC cells. Therefore, we hypothesized that this unknown function gene AK096649 may play an important role in HCC metastasis and recurrence. This project aims to detect the expression level of AK096649 in a larger HCC samples and analyze its relationship with metastasis and recurrence of HCC. Moreover, the effect of AK096649 on the biological behavior of HCC cells will be studied at cellular and animal level. And further experiments will be performed to explore the proteins interacting with AK096649 and their effects on the HCC cells signaling pathways. This project will lay the foundation for elucidating the molecular mechanism of AK096649 promoting the proliferation, metastasis and recurrence of HCC. The successful completion of this project has great significance for understanding the molecular mechanism of HCC metastasis and recurrence and finding novel targets for the treatment of HCC.

肝细胞癌（肝癌）发病率高、转移复发率高、生存期短，分子机制尚不清楚。寻找肝癌相关基因及其在转移复发中的分子机制意义重大。研究表明，lncRNA与肿瘤转移复发密切相关。我们前期发现lncRNA AK096649在76.1%肝癌组织中表达上调，其高表达与肝癌转移复发及低生存期等有相关性；RNA干扰技术沉默AK096649表达可显著降低肝癌细胞的侵袭、迁移能力。因此，我们假设未知功能基因AK096649可能在肝癌转移复发中起到重要作用。本项目拟在更大规模肝癌样本中检测AK096649表达水平，并分析其与肝癌转移复发的关系；此外，在细胞和动物水平研究AK096649对肝癌细胞生物学行为的影响；鉴定AK096649相互作用蛋白及其对肝癌细胞信号通路的影响，为阐明AK096649促进肝癌细胞增殖、转移复发的分子机制奠定基础。本项目顺利完成对于理解肝癌转移复发的分子机制和发现肝癌治疗新靶点具有重要意义。

肝细胞癌（肝癌）是肝脏恶性肿瘤最常见类型，然其分子机制尚不清楚。研究表明，肝癌的发生、发展与一些长链非编码RNA（lncRNA）的异常表达密切有关。项目组利用表达谱基因芯片检测了8例肝癌组织（未复发组，易复发组肝癌组织各4例）中lncRNA表达谱分析获得30个表达上调候选lncRNAs。结合文献资料及和生物信息学技术分析，同期对该30个中最为感兴趣的lncRNAs（如：lncRNA AK096649、lncRNA 00152、lncRNA AK075442等）进行研究，在临床肝癌组织样本验证的结果提示与高通量基因芯片一致，并扩大样本检测与临床病理特征变量统计分析，以及细胞学表型实验平行系列研究时，发现lncRNA AK096649（lnc-AK096649）和lncRNA 00152（LINC00152）影响肝癌细胞表型更为明显，后续的研究更多地偏向于这两个lncRNAs。在体外敲低肝癌细胞lnc-AK096649、或LINC00152表达后，均可明显降低肝癌细胞增殖、迁移和侵袭能力等。然后，我们通过原位杂交技术及细胞RNA核质分离分析发现lnc-AK096649主要定位在细胞浆中。因此我们推测lnc-AK096649可能通过竞争性内源RNA（ceRNA）机制在肝癌的进程中发挥作用。通过生物信息学预测得到lnc-AK096649与hsa-miR-30a-3P可能通过ceRNA机制在肝癌的进程中发挥作用。并通过敲低肝癌细胞中lnc-AK096649的表达后初步发现hsa-miR-30a-3P表达明显上调。进一步，我们发现与相应的肝癌癌旁组织相比，lnc-AK096649在癌组织中呈高表达。其表达水平与肝癌的肿瘤大小、巴塞罗那分期、微血管侵犯、肿瘤分化、早期复发、血清甲胎蛋白及术后生存预后等有相关性（P < 0.05）。通过双荧光素酶报告分析证实了LINC00152通过吸附miR-139作为竞争性RNA。同时，在肝癌组织样本中也发现了LINC00152和miR-139之间的表达存在负相关性。此外，通过双荧光素酶报告分析及相关挽救实验证实了PIK3CA是肝癌miR-139的靶基因，且miR-139通过负向靶向PIK3CA的PI3K/Akt/mTOR途径发挥调节作用。我们的研究证实了LINC00152可通过miR-139激活PI3K/Akt/mTOR通路促进肝癌的发生。