复发性肝癌克隆型特异性miRNAs标签及对靶基因的调控和应用

Supported by the last National Natural Science Foundation of China, we put forward a new concept of six molecular subtypes of clonal origins of postoperative recurrent hepatocellular carcinoma (RHCC).Briefly,PHCC derived from monoclonal model is considered to be derived from intrahepatic residual cancer cells of the last resected tumor. Clinically, more attention should be paied on the comprehensive treatment for patients with monoclonal RHCC,including, interferon-based therapy, ablation therapy as well as transhepatic arterial chemoembolization for anti-tumor metastasis.Whereas RHCC of polyclonal origin means in nature a newborn of primary tumor.The first choice of treatment for patients with polyclonal RHCC is of still surgical resection,and their opstoperative prognoses are also better than the patients with RHCC from monoclonal origin. .However, it still remains a hot issue in looking for more applied molecular diagnostic markers and detection techniques for determing the patterns of clonal origin of RHCC in clinical lab. Recently,by using quantitative reverse transcriptase PCR (qRT-PCR),we find that let-7g, a microRNA(miRNA), shows a significantly differential expression in paired primary and RHCC tissues.No similar researches on the relationship between miRNA expression profiles and clonal models of RHCC have ever been reported before. .Therefore, for objective evaluation of different clonal patterns of RHCC, we plan using the miRNA chip technology for screening,validation and optimization of clonotype-specific and sensitive miRNA differential expression profiles between primary and RHCC, which are further verified in 60 paired RHCC tissues. We also design to predict the target genes of key miRNAs by public microRNA target prediction databases,and study the functional regulations of key miRNAs to their target genes, including the changes in proliferation,invasion and apoptosis in human hepatoma cell lines of MHCC97-L and MHCC97-H,which are transfected with targeted miRNAs and shRNA based on lentiviral vectors,including let-7g,and the tumorigenic ability was also tested by the tumor formation in nude mice after the vector transfection. We aim to provide theoretical basis and diagnostic tool for clinic to correctly diagnose clonal origins, establish individualized treatment,as well as to evaluate the long-term survival for patients with RHCC.

我们曾研究提出复发性肝癌（RHCC）具有单克隆和多克隆两种起源模式特征，用以指导临床个体化治疗和判断预后，但目前仍缺乏RHCC克隆分型适用的分子标志物及发生机制的研究。我们在近期研究中发现，let-7g在肝癌与配对RHCC组织之间呈显著差异表达。为此，我们拟采用miRNAs芯片技术，扩大优化不同克隆型RHCC差异表达的miRNAs，在60对原发和RHCC配对病例中进行验证，建立起评估RHCC克隆型与发生风险的miRNAs检测谱系及临床实验室适用性检测技术流程；通过靶基因数据库，预测并验证目的miRNAs的靶基因，构建携带目的miRNAs和shRNA的慢病毒载体，检测人肝癌细胞系感染后在靶基因功能调控、癌细胞的增殖、侵袭和凋亡及裸鼠成瘤能力上的变化特点，阐明目的miRNAs在RHCC克隆型发生中的作用机制，为临床评估RHCC克隆类型、判断预后及制定个体化治疗策略提供理论依据和有效的诊断方法。

本课题的目的是拟从不同复发间期复发性肝细胞癌（RHCC）组织中筛选出差异表达的miRNA，并验证其细胞功能，最终提出miRNA谱在评估RHCC发生风险中的意义。在检测60对RHCC基因组微卫星表达差异的基础上，根据克隆起源模式（多中心/MO vs.单中心/IM）及复发时间（<2年：短期复发/>2年：远期复发）分组，随机选取其中8对16例次RHCC制备miRNA芯片进行表达谱检测，筛选出5个（hsa-miR-133a, hsa-miR-4251, hsa-miR-4279, hsa-miR-483-5p, hsa-miR-642b-3p）与肝内转移导致的复发类型及复发时间风险预警相关的miRNAs；重点选取差异表达显著的miR-483-5p与靶基因ALCAM之间的调控机制进行验证和研究。细胞功能实验显示，肝癌细胞中转染miR-483-5p的mimics或inhibitor分别可上调或下调miR-483-5p的表达水平；Transwell迁移及侵袭实验显示，两种HCC细胞系mimics处理组的穿过细胞数量明显增加，经计数显示差异显著（P<0.05，P<0.01），提示上调miR-483-5p可明显提高肝癌细胞的迁移及侵袭能力，inhibitor组的迁移及侵袭能力明显降低（P<0.01）；基因功能干扰实验证实，miR-483-5p是通过下调靶基因ALCAM的表达从而产生促肿瘤细胞迁移及侵袭的作用。在荧光素酶报告载体实验中，我们进一步证实了miR-483-5p可以直接结合在ALCAM的3’-UTR区，如果将3’-UTR区内的识别位点进行突变，则突变型ALCAM对miR-483-5p的诱导无应答。为进一步验证miR-483-5p是通过下调ALCAM表达从而产生促肿瘤细胞迁移及侵袭的作用，我们进行了基因功能干扰实验，将ALCAM的siRNAs感染HCC细胞株SK-Hep1和SMMC-7721，Transwell实验发现干扰后的肝癌细胞的迁移及侵袭能力显著增强，这一结果与肝癌细胞转染miR-483-5p后观察到的现象一致，证实两者之间存在负向调控机制；在功能回复实验中，Transwell实验显示，miR-483-5p促进肝癌细胞迁移及侵袭的能力在ALCAM再次引入细胞系后被翻转，表明miR-483-5p是通过直接作用于ALCAM的方式促进肝癌细胞的迁移及侵袭能力提高，有预警作用。