LncRNA TMEM75功能鉴定及其对原癌lncRNA PVT1的转录调控机制研究

Among more than 50,000 human lncRNAs, only a few of lncRNAs have been elucidated for their biological functions and their involvement in human diseases. Using RNAseq Next Generation Sequencing technology, we detected the lncRNA expression profiling of 21 tissues including normal liver, hepatocellular carcinoma (HCC) and portal vein tumor thrombus (PVTT). We found that lncRNA TMEM75 is the most significantly, gradually up-regulated lncRNA in the three groups of tissues. LncRNA TMEM75 locates in the chromosome 8q24 region, a well-known cancer susceptibility region identified by several genome-wide association studies. Expression of lncRNA TMEM75 was positively correlated with the expression of its host gene lncRNA PVT1 in HCC and PVTT tissues. After expression of lncRNA TMEM75 was silenced in HCC cells, expression and the promoter H3K4me3 modification levels of onco-lncRNA PVT1 were significantly suppressed. RNA-pulldown and RNA-IP assays identified and verified that lncRNA TMEM75 can interact with WDR5. Additionally, there is one CpG island in the PVT1 promoter region. Based on the aforementioned results, we aimed to reveal the biological role of lncRNA TMEM75 in HCC and the exact molecular mechanisms on how it regulates the trancription of lncRNA PVT1 in HCC cells. The results of the study will reveal a new mode of transcriptional regulation between different lncRNAs with TMEM75 as an expamle. Also, these results would lay the groundwork for further understanding of the importance of the complex lncRNA regulatory network in human diseases.

在人类逾5万种lncRNA中，只有少数分子的功能及其与人类疾病的关系已被阐明。我们通过RNAseq检测了21例配对的正常肝、肝癌和门静脉癌栓组织的lncRNA表达谱后，发现在三种组织中呈依次表达上调最显著的lncRNA为TMEM75。后继研究显示：TMEM75位于染色体8q24肿瘤易感区段，与其宿主基因lncRNA PVT1的表达呈显著正相关；基因沉默TMEM75后，细胞中癌基因PVT1表达及其启动子区H3K4me3修饰均被显著抑制；RNA-pulldown和RNA-IP实验发现并验证了TMEM75可与WDR5蛋白相互作用；PVT1启动子区存在1个CpG岛。基于此，本项目拟深入研究TMEM75在肝癌中的功能及其对原癌lncRNA PVT1的转录调控机制。研究结果将以TMEM75为例揭示lncRNA之间的转录调控新模式，这也为深入理解复杂lncRNA调控网络在人类疾病中的重要作用打下基础。

在人类逾5万种lncRNA中，只有少数分子的功能及其与人类疾病的关系已被阐明。本项目通过RNAseq检测了21例配对的正常肝、肝癌和门静脉癌栓组织的lncRNA表达谱后，发现在三种组织中呈依次表达上调最显著的lncRNA为TMEM75。我们研究发现，lncRNA TMEM75与其宿主基因lncRNA PVT1的表达呈显著正相关，抑制肝癌细胞中TMEM75表达可显著降低lncRNA PVT1启动子区H3K4me3修饰水平和RNA Pol II结合能力。深入研究显示， lncRNA TMEM75可与WDR5蛋白相互作用，进而募集组蛋白赖氨酸甲基转移酶MLL1至PVT1启动子并继而调控该区域H3K4me3修饰水平。本项目中，我们建立了shRNA稳定基因沉默TMEM75基因(TMEM75-/-)和稳定过表达TMEM75(TMEM75+)的HepG2和Sk-Hep1肝癌细胞系。我们利用上述TMEM75-/-和TMEM75+肝癌细胞系研究发现，lncRNA TMEM75显著促进肝癌细胞增殖、克隆形成能力和小鼠皮下移植瘤生长；裸鼠尾静脉肿瘤细胞注射实验结果显示，lncRNA TMEM75显著促进肝癌细胞的侵袭转移能力。本项目研究结果揭示了lncRNA之间的转录调控新模式，这也为深入理解恶性肿瘤的表观遗传学发病机制打下坚实的基础。