TARBP2和m6A依赖的外泌体miRNAs筛选、鉴定及作为肝癌转移标志物的评价

miRNAs play a critical role in tumor initiation and progression of hepatocellular carcinoma (HCC). The alterations of TARBP2 (the core component for miRNA biogenesis) contribute to miRNA dysregulation, affecting tumor growth, invasion and metastasis in several cancers. Recent findings reveal that the m6A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis，and METTL14 suppresses the metastatic potential of HCC by modulating m6A-dependent pri-miRNA processing, and HCC-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic miRNAs. Our previous studies found there was a C430G mutation of TARBP2 in normal hepatocyte and HCC cell lines such as L02, HepG2, Hep3B, Bel-7402 and Qgy-7701, but not in metastatic HCC cell lines (HCC-LM9 and MHCC-97L). Most important, we also investigated that TARBP2 C430G mutation caused the repression of TARBP2 mRNA and protein expression. GSE data showed that the mRNA expression of TARBP2 in patients with metastatic HCC significantly increased compared with patients with primary HCC. Thus, we hypothesize that TARBP2 C430G mutation and m6A may induce tumor invasion and change the expression level of exosomal miRNAs. To verify the hypothesis, we will first analyze exosomal miRNA profiles of different HCC cell lines with or without TARBP2 C430G mutation, and with METTL3/14 knockdown or overexpression using miRNA expression microarray, m6A-seq will be next utilized to obtain m6A profiling of pri-miRNAs, also the exosomal miRNA in serum of patients with primary HCC or metastatic HCC will be determined by droplet digital PCR. These results will provide novel predictive biomarkers for hepatocellular carcinoma metastasis.

miRNA与肝癌发生发展相关。最新发现：① miRNA合成关键蛋白TARBP2异常与多种肿瘤的侵袭转移相关；② m6A影响pri-miRNA成熟，与肝癌侵袭转移相关；③高转移性肝癌细胞外泌体通过miRNA诱导非转移性肝细胞侵袭迁移。我们发现：TARBP2基因C430G突变仅存在于正常肝细胞和非转移性肝癌细胞，并导致mRNA和蛋白表达明显下降；转移性肝癌患者TARBP2较非转移性明显升高。据此假设：TARBP2和m6A依赖的外泌体miRNA调控肝癌的侵袭转移。本项目拟用miRNA芯片筛选TARBP2野生/突变型、METTL3/14敲减/过表达型的肝癌细胞外泌体差异miRNA；m6A-seq分析pri-miRNA的m6A；实时无标记细胞分析系统观察外泌体受体细胞增殖迁移，及其在裸鼠体内成瘤性、转移性；ddPCR检测血清外泌体候选miRNA。研究将为肝癌转移的精准诊断和治疗提供新的分子标志物。

肝细胞肝癌（hepatocellular carcinoma，HCC）是世界范围内高发生率高死亡率的恶性肿瘤之一。除了HBV感染之外，microRNA, N6-甲基腺嘌呤（N6-methyladenosine，m6A）修饰等表观遗传因素也参与了其发生发展。TARBP2（Transactivation Response Element RNA-binding Protein, TARBP2）是 microRNA在生成和成熟过程中的一种关键蛋白。现有研究表明TARBP2和m6A对多种恶性肿瘤的发生和发展具有一定调控作用；但TARBP2和m6A在在HCC中的作用和分子机制尚未阐明。因此，本项目首先利用不同的肝癌细胞系、病人组织标本和外周血标本进行研究，通过表达、敲减、回补实验，证实TARBP2-miR-145-SERPINE1轴在肝癌进展中的作用。同时，针对乙肝病毒感染是我国肝癌的主要风险因素，建立了高灵敏特异性的HBV-DNA和cccDNA的微滴式数字PCR检测方法，可以实验单细胞和不同的临床样本检测，发现联合检测血清中HBV cccDNA拷贝数和HBV DNA拷贝数可以作为HCC 早期诊断的指标。我们还利用液质联用技术检测组织中m6A修饰水平，发现m6A在肝癌组织中升高，METTL3、METTL14和FTO在肝癌组织中表达下降，且均与m6A水平呈现负相关。进一步的FTO过表达、敲减和回补实验，证实m6A修饰可影响肝癌的发生发展。