RNA甲基化酶METTL14介导的lncRNA-m6A修饰在肝癌干细胞中的作用及机制研究

Hepatocellular carcinoma (HCC) continues to be one of the most common malignant cancer in our country, and the primary cause for the poor prognosis of HCC is the high rates of recurrence and metastasis. Some researches consider that the existence of cancer stem cells results in the high rates of recurrence and metastasis of HCC, so the study of signal pathway of the stem cell in liver cancer is salutary for the early diagnosis and treatment of HCC patients. Accumulating evidence demonstrates m6A (N-6-methyladenosine) modification has an important role in regulating gene expression, such as organ development and disease. Our preliminary study proves that METTL14 is the key catalytic gene for abnormal m6A modification in HCC and is highly associated with metastasis and stem features of HCC. On the other side, long noncoding RNA (lncRNA), an important regulatory factor in physical and pathological status, is believed to be associated with m6A modification and the pathway of the stem cells. On this basis, we plan to use some technologies, such as RNA-seq and MeRIP-seq, to screen the m6A modified lncRNAs which is implicated in the pathway of stem cells, and deeply investigate the regulatory mechanisms of METTL14 in this pathway. Finally, we will test the therapeutic potency of this gene by adding METTL14 mimics to HCC cells. Together, our objective here is to study the function and mechanism of m6A modification and lncRNA in the pathway of stem cells, and offer possible targets for HCC therapy and prediction.

肝癌是我国最常见的恶性肿瘤之一，高转移高复发率是其预后不佳的主要原因。研究显示肝癌干细胞的存在导致了肝癌的高转移和复发，故研究干细胞相关通路可有助于肝癌的早期诊断和治疗。近年研究表明m6A修饰可参与多种通路的调控，在胚胎发育及疾病发生等过程中有着重要的功能。本课题组的前期研究显示，METTL14是肝癌中m6A修饰紊乱的关键调控基因，其介导的m6A修饰与肝癌转移及干性特征密切相关；并且在生理及各种疾病状态下都有重要调控功能的一类分子—lncRNA，也被认为可参与肿瘤干性通路以及被m6A修饰。故本项目拟利用MeRIP-seq等技术筛选肝癌干性通路中受到m6A调控的lncRNA，进一步研究METTL14对靶标lncRNA及干性通路的调控机制，及将METTL14作为靶点治疗肝癌的可能性，深入探讨m6A修饰及lncRNA在肝癌干性通路中的功能和作用，为肝癌的诊断治疗提供新的候选标志物和药物作用靶点。

RNA甲基转移酶METTL14在肝癌的发生发展过程中起着重要作用，其通过诱导RNA甲基化修饰，对下游靶RNA分子的表达、功能等起着重要而又复杂的调控作用。在本研究的资助下，我们重点研究了METTL14通过调节下游靶RNA分子的m6A修饰，对下游靶分子的可变剪接、稳定性的影响，及进而对肝癌肿瘤生物学行为的影响。发现METTL14通过诱导CLSTN1 mRNA的m6A修饰，促进剪切因子MBNL3与CLSTN1 mRNA的结合，进而促进MBNL3对CLSTN1 mRNA第11号外显子的可变剪接，即促进第号外显子的保留，上调含有第11号外显子的较长的CLSTN1转录本（CLSTN1-L）的表达，下调不含第11号外显子的较短的CLSTN1转录本（CLSTN1-S）的表达。功能学实验发现CLSTN1-L、CLSTN1-S发挥着相反的肿瘤生物学功能，即CLSTN1-L促进肝癌细胞的增值、迁移、侵袭、干性，抑制肝癌细胞的凋亡；CLSTN1-S抑制肝癌细胞的增值、迁移、侵袭、干性，促进肝癌细胞的凋亡。此外，我们还鉴定出一条受METTL14调控的长链非编码RNA (lncRNA) 分子-RAB11B-AS1，发现METTL14诱导RAB11B-AS1的m6A修饰，m6A修饰使RAB11B-AS1的RNA稳定性降低。因此METTL14下调RAB11B-AS1的表达。METTL14与RAB11B-AS1的表达在肝癌组织中成负相关。功能学实验发现RAB11B-AS1抑制肝癌细胞的增值、迁移、侵袭、干性，促进肝癌细胞的凋亡。因此本研究揭示了METTL14通过调节RNA甲基化修饰，改变下游靶分子的可变剪接、稳定性，在肝癌中发挥着复杂的调控作用。为完整认识METTL14及其诱导的RNA m6A修饰提供了新视角，也为肝癌诊治提供了新的候选靶点。