缺氧微环境下血管内皮细胞相关microRNA对肿瘤血管生成的作用及机制研究

Hypoxic micro-environment is closely related to angiogenesis, progression and metastasis in malignant tumor. Several studies have identi?ed miRNAs which are regulated by hypoxia. Whereas the research advancement and our primary experiment results, we analyzed and identified miRNA profile under hypoxia inducement (DFO-treatment) in HUVEC by using miRNA array, among these miRNAs, relative expression of 17 miRNA were further performed by qRT-PCR. Our primary results indicated miR-29a and miR-125a-5p promoted and inhibited cell proliferation in HUVEC respectively. Basing the results, we hypothesize that miR-29a and miR-125a-5p affect tumor angiogenesis by regulating key signal molecular in angiogenesis. Around this hypothesis, this project will include following researches: First，we will analyze the effect of the two miRNAs on proliferation, migration, apoptotis and tube network formation of HUVEC in vitro，and testify the correlations between the miRNAs and tumor angiogenesis in 50 specimens from the patients with hepatocellular carcinoma. Next, we will identify the direct target of the miRNAs by using qRT-PCR、western blot and Luciferase reporter assay, and verify the correlation between the miRNAs and varied pro-/anti-angiogenestic factors by using gene chip. Furthermore, we establish nude mouse xenograft models of liver carcinoma with HepG2 cell with lentivirus vector-transfected and expressing the miRNAs or anti-miRNAs(miR-29a or miR-125a-5p), and validate the effect of miRNAs on tumor angiogenesis in vivo. The research findings possibly offer a new and effective target for treating recurrence and metastasis of hepatocellular carcinoma.

缺氧微环境与肿瘤血管生成及肿瘤转移密切相关。我们前期经芯片筛选、qRT-PCR验证获得了缺氧诱导下内皮细胞相关miRNA，预实验显示miR-29a及miR-125a-5p分别促进和抑制了血管内皮细胞增生。基于前期研究结果，本课题将围绕"miR-29a、miR-125a-5p调控血管生成关键分子表达，影响肿瘤血管生成"这一假说，开展以下研究：首先，分析所候选的miRNA对肝癌血管内皮细胞增殖、凋亡、迁移、管状结构形成的影响；利用临床肝癌标本检测miRNA表达与血管生成的相关性；其次，通过PCR、western blot、荧光素酶报告载体等方法验证miRNA的直接靶基因；生物芯片技术分析miRNA与重要血管生成促进或抑制因子之间的关系；最后，建立裸鼠肝癌移植瘤模型，用携带候选miRNA质粒的重组慢病毒载体验证miRNA体内抗肿瘤血管作用。该研究的成果将为肝癌复发与转移的治疗提供新的靶点。

在前期基因芯片筛选缺氧诱导内皮细胞差异表达miRNA，并经qRT-PCR定量检测基础上，验证 miR-29a是否能调控人血管内皮细胞血管生成的能力。结果提示miR-29a在血管内皮细胞中高表达，低氧状况下诱导了miR-29a的表达上调。外源性agomiR-29a促进内皮细胞生存或增殖，促进了内皮细胞管状结构形成；体内Matrigel plug方法检测结果显示给予29a-AgomiR过表达miR-29a组小鼠比对照组新生血管数量明显增加。HBP1是miR-29a的靶基因。研究结果表明miR-29a对人血管内皮细胞的细胞周期、增值及血管生成特性的作用可能通过靶向HBP1的转录后调 控。近年来，相关研究发现lncRNA在恶性肿瘤发生和发展中起着重要作用。作为ncRNA中的新角色lncRNA，在癌症中起调控作用。因此，课题组对肝细胞肝癌(HCC)及甲状腺乳头状癌(PTC)组织进行RNA -seq，通过比对癌组织与癌旁组织表达谱的差异，更精确地挖掘出有潜在价值的突变体及非编码RNA。我们发现了69 个在HCC癌/癌旁组织中呈显著性差异表达的lncRNA。在21对肝癌及癌旁组织中利用real-time PCR对差异最显著的26个lncRNA进行了表达 量的验证，发现17个lncRNA的表达差异具有统计学意义。同时，我们还对差异表达基因进行了分析，发现了217个具有显著性差异表达的基因。同样，我们对差异最显著的27个基因在21对肝癌及癌旁组织中进行real-time PCR验证，发现17个基因表达差异具有统计学意义。在检出的差异表达lncRNA中，LNC-1，4可能在肝癌细胞中发挥着抑制细胞增殖的功能。另外我们检测和验证测序组和验证组PTC相对于癌旁组织中lncRNA表达谱的变化。分析这些差异基因的临床特征分布，筛选临床特征相关性lncRNAs。计算差异lncRNA与差异蛋白编码基因的相关关系，同时分析这些差异基因参与的信号通路，通过分析共表达的蛋白编码基因解析lncRNA的潜在功能。最后检PTC癌组织中BRAF V600E突变状态，分析并构建BRAF 突变特异性的PTC中lncRNA表达亚谱。甲状腺乳头状癌相关的lncRNAs：NONHSAG051968、NONHSAT076747 and NONHSAT122730可能是其潜在的诊断和治疗的靶点。